Human TSC403 gene and human ING1L gene

ABSTRACT

This invention provides the TSC403 gene having a nucleotide sequence coding for the amino acid sequence of SEQ ID NO:1, which is a novel gene of great utility particularly in the field of research, diagnosis, therapy, etc. for cancer of the lung, among other diseases.  
     In addition, this invention provides the human ING1L gene comprising a nucleotide sequence coding for the amino acid sequence of SEQ ID NO:4, which is a novel human gene useful for regulating the cell cycle, inhibiting or activating cell proliferation, studies on metabolic aging or apoptosis of cells, pathological exploration, diagnosis and therapy of cancer and other diseases, and screening for the development of new drugs.

TECHNICAL FIELD

[0001] The invention relates to a gene of use as an index in theprophylaxis, diagnosis and therapy of human diseases and moreparticularly to a novel lung-specific human gene which is homologous tohuman 1 amp-1 and -2 [lysosomal membrane glycoprotein; Saito, O. et al.,J. Biol. Chem., 267, 5700-5711 (1992); Sawada, R. et al., J. Biol.Chem., 268, 12675-12681 (1993); Sawada, R. et al., J. Biol. Chem., 269,1425-1431 (1994)] and suspected to act as an oncogene.

[0002] The invention further relates to a novel human gene which isanalogous to the rat, mouse, yeast, nematode, known human and othergenes and, through the cDNA analysis, chromosome mapping and functionalanalysis of its cDNA, can be utilized in gene diagnosis and for thedevelopment of new therapeutic drugs.

[0003] In addition, the invention relates to novel proteins encoded bysaid genes and to specific antibodies thereto.

BACKGROUND ART

[0004] The genetic information in organisms is accumulated as arrays(DNA) of four kinds of bases, viz. A, C, G and T, in the cell nucleus,and this genetic information is conserved for maintenance of lineage andontogenesis. In a human being, the number of such bases is said to beapproximately three-billion (3×10⁹) and it is estimated that thispopulation includes 50-100 thousand genes. The genetic information isinvolved in the maintenance of vital phenomena through the creation ofregulatory proteins, structural proteins, enzymes, etc. along the flowof transcription of mRNA from genes (DNA) and ensuing translation intoproteins.

[0005] It is generally acknowledged that any abnormality of the aboveflow from a gene to its translation product protein leads to an error ofthe life maintenance system inclusive of the proliferation anddifferentiation of cells, and can be causes of various diseases. Theresults of gene analyses made to this day suggest that the genes ofvarious receptors, such as the insulin, LDL and other receptors, andthose of metabolic enzymes associated with the growth anddifferentiation of cells, such as protease, ATPase, superoxidedismutase, etc., are considered to be useful tools for the developmentof pharmaceuticals.

[0006] However, the analysis of human genes and the study of theirfunctions and relationships to various diseases are still in theinchoate stage and much remains to be known. Therefore, analysis of newgenes, analytical explorations into the functions and relationships todiseases of such genes, and studies for the establishment of genediagnostics exploiting the genes so analyzed, and pharmaceuticalapplication studies on such genes are subjects of immense interest tothis industry.

[0007] Meanwhile, carcinoma of the pancreas is one of the malignanttumors of the digestive system with the poorest prognosis, rankingfourth and fifth on the list of causes for cancer-related death in Japanand Western counties, respectively (Poston, J. G., et al., Gut., 32,800-812 (1991)). The most important goal in cancer research is toidentify changes in the genes in the early phase of oncogenesis.Identification of such changes should lead to the development of genetictools for early diagnosis and novel therapeutic modalities for effectivetreatment of this lethal disease.

[0008] Elucidation of the physiological roles of such genes and theresulting information are important to the explication of the mechanismsof genesis and onset of neoplastic diseases, and have been demanded notonly in the field of fundamental scientific research but also from thestandpoint of characterization and treatment of malignant tumors in thepharmaceutical field.

DISCLOSURE OF INVENTION

[0009] Thus, assuming that a novel human gene be provided, itsexpression levels in various cells as well as its structure andfunctions could be elucidated and through analysis of expressionproducts of the gene, the clarification of pathology, diagnosis andtherapy of the diseases associated with the gene, such as hereditarydiseases and cancers, would become feasible. The object of the inventionis to provide such novel human genes.

[0010] With the above object in mind, the inventors did intensiveresearch as described below. Thus, to begin with, the inventorssynthesized cDNAs from the mRNAs extracted from various human tissuessuch as human fetal brain, adult blood vessel and placenta, cloned theminto vectors to construct libraries, cultured Escherichia coli cellstransformed with each library on agar medium, picked up transformantcolonies at random and transferred them to microtiter plates to prepareand register E. coli clones containing various human genes. Then, eachof these clones was cultured, the DNA extracted and purified, and usingthe cDNA thus obtained as a template, an amplification reaction withchain termination specific to said 4 bases is carried out by the deoxyterminator method, and using an automatic DNA sequencer, the sequence ofabout 400 nucleotides from the 5′ end of the human gene in eachregistered clone was determined. Based on the thus-obtained nucleotidesequence information on human genes, novel family genes similar to theknown bacterial, yeast, nematoid, murine, human and other animal andplant genes were explored. The above technology for cDNA analysis isdescribed in detail in the report of Fujiwara et al. [Fujiwara, Tsutomu,Saibo Kogaku (Cell Engineering), 14, 645-654 (1995)].

[0011] As a result, among the cDNA clones picked up arbitrarily from thehuman fetal brain cDNA library, the inventors found a clone harboring anovel gene which codes for an amino acid sequence having high homologyto p33^(ING1) which is considered to be a cancer-suppressive protein[GenBank A. C. No. AF001954, Garkavetsev, et al., Nature, Genet., 14,415-420(1996); Garkavetsev, et al., Mol. Cell. Biol., 17, 2014-2019(1997); rewrote-GenBank A. C. No. AF044076]. This invention has beendeveloped on the basis of the above finding.

[0012] Furthermore, for the purpose of providing said informationdemanded by the industry, in particular a gene coding for a novelprotein having homology to lamp-1 gene and lamp-2 gene, the inventorsmade an intensive exploration into the genes derived from various humantissues and succeeded in isolating and characterizing a novellung-specific gene matching for the above purpose. This invention hasbeen developed on the basis of the above finding.

[0013] Thus, in the first place, the invention provides a genecontaining a nucleotide sequence coding for the amino acid sequence ofSEQ ID NO:1 (hereinafter referred to TSC403 gene), in particular saidgene which is a human gene.

[0014] In addition, the invention provides a novel protein encoded bysaid TSC403 gene (hereinafter referred to as TSC403 protein) and anantibody having a binding affinity for said protein.

[0015] Further, the invention provides a TSC403 gene which is any one ofthe following polynucleotides (a), (b) and (c), particularly said genewhich is a human gene.

[0016] (a) a polynucleotide containing the nucleotide sequence of SEQ IDNO:2 or a complementary chain thereto;

[0017] (b) a polynucleotide which hybridizes to a DNA having thenucleotide sequence of SEQ ID NO:2 under stringent conditions; and

[0018] (c) a polynucleotide having at least 95% homology to apolynucleotide coding for a polypeptide containing the amino acidsequence of SEQ ID NO:1

[0019] The invention further provides a TSC403 gene having thenucleotide sequence of SEQ ID NO:3.

[0020] The invention further provides an oligonucleotide having asequence consisting of at least 15 consecutive nucleotides in thenucleotide sequence of SEQ ID NO:2 and a DNA fragment for use as aspecific probe or primer for detecting genes having said oligonucleotidesequence.

[0021] Furthermore, in accordance with the invention, there is provideda human gene (hereinafter referred to as human ING1L gene) containing anucleotide sequence coding for the amino acid sequence of SEQ ID NO:4.

[0022] This invention further provides a protein (hereinafter referredto as human ING1L protein) which is encoded by said human ING1L gene andan antibody binding said protein.

[0023] Further provided in accordance with this invention is a humanING1L gene comprising any one of the following polynucleotides (a), (b)and (c).

[0024] (a) a polynucleotide containing the nucleotide sequence of SEQ IDNO:5;

[0025] (b) a polynucleotide containing a nucleotide sequence whichhybridizes to a DNA having the nucleotide sequence of SEQ ID NO:5 understringent conditions; and

[0026] (c) a polynucleotide having at least 95% homology to apolynucleotide coding for a polypeptide containing the amino acidsequence of SEQ ID NO:4.

[0027] Further provided in accordance with this invention is a humanING1L gene having the nucleotide sequence of SEQ ID NO:6.

[0028] In addition, according to the invention, there are provided anoligonucleotide having a sequence consisting of at least 15 consecutivenucleotides in the nucleotide sequence of SEQ ID NO:5 and a DNA fragmentfor use as a specific probe or primer for detecting genes having saidoligonucleotide sequence.

[0029] Representation of amino acids, peptides, nucleotide sequences,nucleotides, etc. by abbreviations in this specification is inconformity with the rules recommended by IUPAC-IUB [IUPAC-IUBCommunication on Biological Nomenclature, Eur. J. Biochem., 138, 9(1984)], “Guideline for Preparation of a Specification or EquivalentReferring to a Nucleotide Sequence and/or an Amino Acid Sequence”(edited by the Patent Office of Japan) and the conventions relating tothe use of codes or symbols in the art.

[0030] The TSC403 gene according to the invention is now described indetail.

[0031] As a specific example of the TSC403 gene according to theinvention, there can be mentioned the gene deduced from the DNA sequenceof a PCR product named “TSC403” as described in the Example whichappears hereinafter. Its nucleotide sequence is presented in SEQ IDNO:3.

[0032] This gene is a human cDNA coding for a novel lung-specificprotein having a sequence of 416 amino acid residues as shown in SEQ IDNO:1 (hereinafter referred to as “TSC403 protein”) and this cDNA has afull length of 3198 nucleotides.

[0033] The TSC403 protein of the invention occurs as an expressionproduct of the gene of the invention. A homology search using FASTAProgram [Person, W. R., et al., Proc. Natl. Acad. Sci., USA, 85,2444-2448 (1988)] against the GenBank/EMBL database revealed that thisgene is homologous to human lamp-1 gene and lamp-2 gene (cf. theliterature cited above).

[0034] In this connection, it is known that said human lamp genes areexpressed at high levels in a highly metastatic colorectal cancer cellline and bind to E-selectin on the vascular endothelial cell. It is,therefore, suspected that these genes are associated with the malignancyof cancers (the literature cited above).

[0035] The TSC403 gene according to the invention is also acancer-related gene, which is expected to find application as a cancermarker.

[0036] Furthermore, the chromosomal locus of this gene of the inventionis 3q27 where chromosomal aberration is detected in various cancers.This fact, even alone, strongly suggests the relation this gene of theinvention has to various neoplastic diseases.

[0037] Furthermore, the TSC403 gene according to the invention was foundto show high expression in various cancer specimens, suggesting itsvalue as a marker for predicting oncogenesis and malignancy.

[0038] Thus, the TSC403 gene or a gene product thereof in accordancewith the invention provides the information or means of immenseimportance to the elucidation, understanding, diagnosis, prophylaxis andtherapy of various neoplastic diseases such as colorectal cancer, cancerof the uterus, cancer of the ovary, cancer of the lung, and cancer ofthe pancreas, among others. Furthermore, this gene of the invention canbe used with advantage in the development of new drugs which wouldinduce expression of the gene for use in the treatment of saidneoplastic diseases.

[0039] In addition, detection of expression of the gene of the inventionor expression of its product in an individual or a given tissue as wellas detection of a mutation (deletion or point mutation) or expressionabnormality of said gene can be exploited to advantage in theexplication and diagnosis of said various neoplastic diseases.

[0040] The human ING1L gene of the invention is now described in detail.

[0041] As a specific example of the human ING1L gene according to theinvention, there can be mentioned the gene deduced from the DNA sequenceharbored by the clone named “GEN-146F11” and described in the Examplewhich appears hereinafter. The nucleotide sequence of this gene ispresented in the SEQUENCE LISTING. Thus, the gene harbored by this clonehas a 840-nucleotide open reading frame (deduced amino acid translatedregion; the sequence is shown in SEQ ID NO:5) which codes for thesequence of 280 amino acid residues as shown in SEQ ID NO:4 in theSEQUENCE LISTING, and the full-length nucleotide sequence of the cDNAclone consists of 1078 nucleotides as shown in SEQ ID NO:6.

[0042] In the above sequence of SEQ ID NO:6, the initiation codon islocated in the position 92-94 and the termination codon in the position932-934. The polyadenylation signal-like sequence (ATTAAA) is located inthe position 1058-1063.

[0043] As mentioned above, the human ING1L gene of the invention hashigh homology to p33^(ING1) and can be utilized in the analysis of humangenes based on its genetic information and studies on the relationshipsof various functions of the genes so analyzed to various diseases andfurther exploited in the gene diagnosis and gene therapy of thegene-related diseases and application studies on the genes in thepharmaceutical field. Thus, the functions of the protein (gene product)encoded by the human ING1L gene of the invention can be predicted fromthose of the known homologous genes, and as the result of provision ofthe gene of the invention, it is now possible to construct a recombinantprotein by cloning the candidate gene in an expression vector andinvestigate its enzymatic activity, binding activity and otherfunctions. Particularly, since the gene of the invention is suspected tofunction as an oncogene, this function can be utilized with advantage inthe development of pharmaceuticals such as anticancer drugs.

[0044] The protein (hereinafter referred to as “human ING1L protein)encoded by the human ING1L gene of the invention has a Zn fingermotif-like sequence in its C-terminal region and this region inparticular is considered to have high homology to said p33^(ING1).

[0045] In this connection, it has been reported that said p33^(ING1) isinactivated in several cancer-derived cell lines including a mammarycancer cell line [the literature cited above]. Moreover, it has recentlybeen demonstrated that said p33^(ING1) is negatively regulating cellproliferation through p53 which is known to be a cancer-suppressive geneproduct [Garkavetsev, et al., Nature, 391, 295-298 (1998)]. Furthermore,in various human neoplastic tissues, the expression level of human ING1Lgene is specifically elevated. From these findings, it is suspected thatthe human ING1L protein is positively modulating cell proliferationthrough its interaction with p53.

[0046] Furthermore, in Northern blot analysis, expression of the humanING1L gene of the invention was found in all the 16 human adult organ-derived tissues tested and enhancement of its expression was noted inseveral neoplastic tissues including colorectal cancer, cancer of theesophagus, cancer of the uterine tube, and stomach cancer as comparedwith the normal tissues. These findings suggest that the gene of theinvention can be used for the diagnosis of neoplastic and other diseasesassociated with it by checking for the expression thereof in varioustissues and, as a corollary, finds application in the screening forantimitotic compounds or anticancer compounds.

[0047] The gene of the invention specifically includes polynucleotidescontaining the nucleotide sequences of SEQ ID NOS:2 and 5 which code forthe amino acid sequences of SEQ ID NOS:1 and 4, respectively,polynucleotides which hybridize to DNAs containing the nucleotidesequences of SEQ ID NOS:2 and 5 under stringent conditions, andpolynucleotides having at least 95% homology to polynucleotide encodingthe amino acid sequences of SEQ ID NOS:1 and 4.

[0048] Therefore, the gene of the invention includes those genes whichencode amino acid sequences corresponding to certain modifications ofthe above-defined amino acid sequences and those genes which have adefined degree of homology to the above-defined nucleotide sequences.

[0049] Thus, the gene of the invention includes, among others, genescontaining nucleotide sequences coding for the amino acid sequencesresulting from the deletion, substitution or addition of one or aplurality of amino acids from, in or to the amino acid sequence of SEQID NO:1 or 4 (i.e. modified amino acid sequences). The gene having anucleotide sequence coding for such a modified amino acid sequence needonly be such that by utilizing it, the gene of the invention coding forthe unmodified amino acid sequence can be detected.

[0050] Incidentally, while such modifications (mutation etc.) of aminoacid sequences may be spontaneous, e.g. mutations and post-translationalmodifications, the modifications can be made artificially as well byutilizing a gene of the natural origin (for example, a specific gene ofthe invention).

[0051] The means for making such artificial modifications includesgenetic engineering techniques such as site-specific (-directed)mutagenesis [Methods in Enzymology, 154: 350, 367-382 (1987); ditto 100:468 (1983); Nucleic Acids Res., 12: 9441 (1984); Zoku Seikagaku JikkenKoza 1 “Idenshi Kenkyuho II” [Experimental Biochemistry Series 1“Methods for Gene Research II” (edited by Japanese Biochemical Society),p105 (1986)], etc. and chemical synthetic techniques such as thephosphotriester method and the phosphoamidate method [J. Am. Chem. Soc.,89: 4801 (1967); ditto 91: 3350 (1968); Science, 150: 178 (1968);Tetrahedron Lett., 22: 1859 (1981); ditto 24: 245 (1983)] as well as asuitable combination of such techniques.

[0052] As one example of the gene according to the invention, the genecomprising a polynucleotide having the nucleotide sequence of SEQ IDNO:2 or 5 or a complementary sequence thereto can be mentioned. Thisnucleotide sequence represents an example of combination of codons foreach amino acid residue of the above amino acid sequence (SEQ ID NO:1 or4). Of course, the gene of the invention is not limited to the abovecombination but the gene having a nucleotide sequence designed byselecting an arbitrary combination of codons for each of said amino acidresidues can be employed. Selection of said codons can be made in theroutine manner. In this selection, the codon frequency of the host to beused may be taken into consideration [Nucleic Acids Res., 9: 43 (1981)].

[0053] Furthermore, while the gene of the invention is shown as thenucleotide sequence of a single-stranded DNA as, for example, shown inSEQ ID NO:3 or 6, the invention of course encompasses a polynucleotidecomprising a nucleotide sequence complementary to such a nucleotidesequence and a component containing both of them as well and, moreover,is not limited to a DNA such as cDNA.

[0054] Furthermore, as mentioned above, the gene of the invention is notlimited to one comprising a polynucleotide having the nucleotidesequence of SEQ ID NO:2 or 5 or a complementary sequence thereto butincludes one comprising a nucleotide sequence having a given degree ofhomology to such a nucleotide sequence. More particularly, there isincluded the gene comprising a polynucleotide having at least 95%homology to a polynucleotide coding for a polypeptide having the aminoacid sequence of SEQ ID NO:1 or 4.

[0055] Moreover, the gene of said nucleotide sequence having a definedhomology includes one that hybridizes to a DNA having the nucleotidesequence of SEQ ID NO:2 or 5 under stringent conditions such as thosedescribed below and does not lose the DNA even when the hybrid is washedunder given conditions.

[0056] As an example, there can be mentioned a gene having a nucleotidesequence which, when hybridized to a DNA having the nucleotide sequenceof SEQ ID NO:2 or 5 in 6×SSC at 65° C. overnight or in 4×SSCsupplemented with 50% formaldehyde at 37° C. overnight and, then, washedin 2×SSC at 65° C. for 30 minutes, will not be disengaged from the DNA.Here, SSC stands for standard saline-citrate buffer (standard salinecitrate; 1×SSC=0.15 M NaCl, 0.015 M sodium citrate). A preferred exampleof said gene is a gene having a nucleotide sequence which, even whenhybridized to a DNA having the nucleotide sequence of SEQ ID NO:2 or 5in 7% polyethylene glycol (PEG)/10% sodium dodecyl sulfate (SDS) at 65°C. overnight and washed in 0.1×SSC/0.1% SDS at 65° C. for 30 minutes,will not be disengaged from the DNA.

[0057] The gene of the invention can be easily produced and acquired bythe standard genetic engineering techniques [Molecular Cloning 2d Ed,Cold Spring Harbor Lab. Press (1989); Zoku Seikagaku Jikken Koza“Idenshi Kenkyuho I, II, III” [New Experimental Biochemistry Series“Methods for Gene Research I, II, III” (edited by Japanese BiochemicalSociety), (1986), etc.] based on the sequence information on thespecific examples shown in SEQ ID NO:3 or 6.

[0058] More particularly, the objective gene can be acquired byconstructing a cDNA library from a suitable source containing the geneof the invention and selecting the desired clone from this cDNA libraryusing a suitable probe or antibody specific to the gene of the inventionin the per se known manner [Proc. Natl. Acad. Sci., USA., 78: 6613(1981); Science, 222: 778 (1983), etc.].

[0059] In the above procedure, the cDNA source includes to various cellsor tissues in which the gene of the invention is expressed and culturedcells derived therefrom. Particularly in the case of the TSC403 gene ofthe invention, lung tissues can be mentioned by way of example.Isolation of the whole RNA from such a source, isolation andpurification of mRNA, synthesis of cDNA, and cloning thereof can all becarried out in the routine manner. cDNA libraries are also commerciallyavailable. In the practice of the invention, such commercial cDNAlibraries, for example those available from Clontech Lab. Inc., can alsobe employed.

[0060] The method of screening for the gene of the invention from a cDNAlibrary is not particularly restricted, either, but a conventionalmethod can be selectively employed. To be specific, selection of a cDNAclone by an immuno screening technique using a specific antibody againstthe protein produced by the cDNA, the plaque hybridization or colonyhybridization technique using a probe having a selective bindingaffinity for the objective DNA sequence, or a combination thereof can bementioned by way of example.

[0061] As to the probe to be used in the above procedure, it isgenerally useful to use a DNA chemically synthesized according to thenucleotide sequence information on the gene of the invention. Of course,it is also possible to use the gene already obtained or a fragmentthereof as said probe.

[0062] The nucleotide sequence which can be used as said probe includesa partial nucleotide sequence corresponding to SEQ ID NO:2 or 5 butconsisting of at least 15 consecutive nucleotides, preferably within therange of 20-30 nucleotides. Moreover, positive clones containing theabove respective sequences can also be utilized as said probe.

[0063] Said screening can be carried out by the procedure which uses, asthe screening probe, a set of sense and antisense primers based on thepartial amino acid sequence information about a natural extract isolatedand purified from a given cell line or tissue.

[0064] Furthermore, said screening can also be carried out by theprotein interaction cloning procedure using the TSC403 protein or humanING1L protein in lieu of said specific antibody.

[0065] In the invention, the expression of mRNA in cells under differentconditions or between a plurality of cell groups can be studied bydirect comparison using the differential display method [Liang, P., etal., Science, 257, 967-971 (1992)].

[0066] In obtaining the gene of the invention, DNA/RNA amplification byPCR [Science, 230, 1350 (1985)] can also be used with advantage.Particularly in case where a full-length cDNA can hardly be obtainedfrom a library, the RACE [rapid amplification of cDNA ends] method[Jikken Igaku (Experimental Medicine), 12(6): 35 (1994)], in particularthe 5′-RACE method [Frohman, M. A., et al., Proc. Natl. Acad. Sci.,USA., 8: 8998 (1988)], can be used with advantage. The primers for usein such PCR methods can be judiciously established according to thesequence information on the gene of the invention and can be synthesizedby the conventional procedure.

[0067] Isolation and purification of the amplified DNA/RNA fragment canbe carried out by the conventional techniques as mentioned hereinbefore,for example by gel electrophoresis.

[0068] The nucleotide sequence of the gene of the invention or any ofvarious DNA fragments thereof can be determined in the routine manner,for example by the dideoxy method [Proc. Natl. Acad. Sci., USA., 74:5463 (1977)], the Maxam-Gilbert method [Methods in Enzymology, 65: 499(1980)] or, more expediently, by means of a commercial sequencing kit.

[0069] With the gene of the invention, the gene product can be producedeasily, on a high production scale, and with good reproducibility by thestandard genetic engineering technology.

[0070] The invention further provides a vector (expression vector)harboring said TSC403 gene or human ING1L gene, host cells transformedby using said vector, and a method of producing TSC403 protein or humanING1L protein which comprises growing said host cells.

[0071] Production of said TSC403 protein and human ING1L protein can becarried out by the standard recombinant DNA technology [Science, 224:1431 (1984): Biochem. Biophys. Res. Comm., 130: 692 (1985): Proc. Natl.Acad. Sci., USA., 80: 5990 (1983), and the reference literature citedhereinabove].

[0072] As said host cells, whichever of prokaryotic cells and eucaryoticcells can be employed. As the prokaryotic host, various procaryoteswhich are commonly employed, such as Escherichia coli and Bacillussubtilis, can be liberally employed. The preferred host cells are thosederived from Escherichia coli, particularly cells of E. coli K12.

[0073] The eucaryotic host cells include cells of vertebrate and yeasts,among others. Among the former cells, the monkey cell line COS [Cell,23: 175 (1981)], Chinese hamster ovarian cells and the dihydrofolatereductase-defective line thereof [Proc. Natl. Acad. Sci., USA., 77: 4216(1980)] can be mentioned as examples. As to the latter cells, cells ofyeasts belonging to the genus Saccharomyces can be mentioned as examplesbut these are not exclusive choices.

[0074] When prokaryotic cells are used as host cells, a vector which canbe replicated in the host cell is selected and, for expression of thegene, an expression plasmid provided with a promoter and the SD(Shine-Dalgarno) sequence upstream of the gene of the invention, as wellas an initiation codon (e.g. ATG) necessary to start protein synthesis,can be employed with advantage. As the vector mentioned above, it isusual to employ an E. coli-derived plasmid, such as pBR322, pBR325,pUC12, pUC13, etc., although these are not exclusive choices and variousknown other vectors may be utilized. As commercial vectors forexpression systems using E. coli, pGEX-4T (Amersham Pharmacia Biotech),pMAL-c2, PMAL-p2 (New England Biolabs), pET21, pET21/lacq (Invitrogen),pBAD/His (Invitrogen), among others, can be mentioned by way of example.

[0075] As the expression vector to be used when cells of a vertebralanimal are employed, usually a vector having a promoter region upstreamof the gene to be expressed, RNA splice sites, polyadenylation site,transcription end sequence, etc. can be mentioned, and where necessary,the vector further has a replication origin. As a specific example ofthe above vector, pSV2dhfr containing an early promoter of SV40 [Mol.Cell. Biol., 1: 854 (1981)], for instance, can be mentioned.

[0076] Aside from the above, various other known commercial vectors canbe used. As commercial vectors which can be used in expression systemsutilizing animal cells, there can be mentioned various vectors availablefor animal cell use, such as pEGFP-N, pEGFP-C (Clontech), pIND(Invitrogen), pcDNA3.1/His (Invitrogen), etc. and vectors available forinsect cell use, such as pFastBacHT (Gibco BRL), pAcGHLT (PharMingen),pAc5/V5-His, pMT/V5-His and pMT/Bip/V5-His (all Invitrogen).

[0077] As a specific example of the expression vector which can be usedwhen yeast cells are used as the host cells, pAM82 having a promoter forthe acid phosphatase gene [Proc. Natl. Acad. Sci., USA., 80: 1 (1983)]can be mentioned. The commercial expresion vectors for yeast cell useinclude to pPICZ (Invitrogen) and pPICZ (Invitrogen).

[0078] The promoter is not particularly restricted, either. When abacterial strain of the genus Escherichia is used as the host,tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter,PL/PR promoter, etc. can be used with advantage. When the host is anorganism of the genus Bacillus, SP01 promoter, SP02 promoter, penPpromoter, etc. are preferred choices. The promoter which can be usedwith advantage when a yeast is used as the host includes pH05 promoter,PGK promoter, GAP promoter and ADH promoter, among others. The preferredpromoter in cases where animal cells are used as said host cellsincludes SV40-derived promoter, retrovirus promoter, metallothioneinpromoter, heat shock promoter, cytomegalovirus promoterandSR promoter,among others.

[0079] As expression vectors for the gene of the invention, theconventional fusion protein expression vector can also be used withadvantage. As an example of the vector of this type, pGEX (Promega) forexpression of a fusion protein with glutathione-S-transferase (GST) canbe mentioned.

[0080] The method of introducing said objective recombinant DNA(expression vector) into the host cell (transformation method) is notparticularly restricted, either, but various standardized methods can beutilized. Culture of the resultant transformant can also be performed inthe routine manner. By such culture, the objective protein encoded bythe gene of the invention is expressed, produced, and accumulated in thetransformant cell or secreted extracellularly or on the cell membrane.

[0081] The medium for said culture can be judiciously selected fromamong the conventional media according to the type of host cellsadopted, and culture can also be carried out under conditions suited forproliferation of the host cells.

[0082] The recombinant protein thus produced can be optionally isolatedand purified by various isolation procedures utilizing its physical,chemical or other properties [Seikagaku (Biochemical) Data Book II,pp.1175-1259, 1st Ed., 1st Impression, Jun. 23, 1980, Tokyo KagakuDojin; Biochemistry, 25(25): 8274 (1986); Eur. J. Biochem., 163: 313(1987); etc.]. The procedures mentioned above specifically include thestandard reconstitution treatment, treatment with a proteinprecipitating agent (salting out), centrifugation, osmotic shock method,sonic disruption, ultrafiltration, various kinds of chromatography, e.g.molecular sieves chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, affinity chromatography,high performance liquid chromatography (HPLC), etc., dialysis, and theircombinations. The particularly preferred procedure is affinitychromatography using a column conjugated with a specific antibodyagainst the TSC403 protein or human ING1L protein according to theinvention.

[0083] The invention further provides the novel TSC403 protein or humanING1L protein obtainable as above and the technology of producing thoseproteins. The protein according to the invention finds application inthe pharmaceutical field as mentioned hereinbefore.

[0084] Moreover, the protein of the invention can be used as animmunogen for construction of a specific antibody against said protein.The component for use here as the antigen may be the protein produced ina large amount by any of said genetic engineering techniques or afragment thereof, and by using such an antigen, the objective antiserum(polyclonal antibody) and monoclonal antibody can be obtained.

[0085] The production technology for such antibodies is well known tothose skilled in the art and the production of antibodies relevant tothe invention can also be made in accordance with such establishedtechnology [Zoku Seikagaku Koza “Men-eki Seikagaku Kenkyuho” (NewImmunobio chemistry Series, “Methods in Immuno biochemistry”), edited byJapanese Biochemical Society (1986), among others].

[0086] For example, the immune animal for use in harvesting theantiserum can be liberally selected from among ordinary animals such asthe rabbit, guinea pig, rat, mouse, chicken, goat and sheep, andimmunization with said antigen and collection of blood can also becarried out in the routine manner.

[0087] Preparation of said monoclonal antibody can also be carried outin the routine manner, i.e. by constructing a fusion cell from theplasma cell (immune cell) of an animal immunized with said immunogen anda plasmocytoma cell, selecting a clone producing the objective antibody,and growing the clone. The immune animal is generally selected inconsideration of its compatibility with the plasmocytoma cell to be usedfor cell fusion and usually the mouse or the rat is used with advantage.Immunization can be carried out in the same manner as in the preparationof said antiserum, and optionally the usual adjuvant can be used incombination with the antigen.

[0088] The plasmocytoma cell to be used for said fusion is notparticularly restricted but may be any of various myeloma cells such asp3 (p3/x63-Ag8) [Nature, 256: 495-497 (1975)], p3-U1 [Current Topics inMicrobiology and Immunology, 81: 1-7 (1978)], NS-1 [Eur. J. Immunol., 6:511-519 (1976)], MPC-11 [Cell, 8: 405-415 (1976)], SP2/0 [Nature, 276:269-271 (1978)], etc., R210 [Nature, 277: 131-133 (1979)], etc. fromrats and cells derived therefrom.

[0089] The fusion between said immune cell and plasmocytoma cell can beeffected in the presence of a conventional fusion promoter, such aspolyethylene glycol (PEG), Sendai virus (HVJ) or the like, in accordancewith a known protocol. Isolation of the objective hybridoma can also becarried out by the known procedure [Meth. in Enzymol., 73: 3(1981); saidZoku Experimental Biochemistry Series; etc.].

[0090] The search for the objective antibody-producing cell line and thepreparation of a monoclonal antibody can also be carried out in theconventional manner. For example, the search for an antibody producingline can be made by various techniques which are generally used fordetection of antibodies, such as ELISA [Meth. in Enzymol., 70: 419-439(1980)], plaque method, spot method, agglutination reaction method,Ouchterlony method, radioimmunoassay, etc., using the protein of theinvention as an antigen.

[0091] Isolation of the antibody of the invention from the hybridomaobtained as above can be carried out by the method which comprisesgrowing the hybridoma in the routine manner and recovering the antibodyas a culture supernatant or the method which comprises administering thehybridoma to a compatible mammal to let it multiply in vivo andrecovering the antibody in the form of an ascite fluid. The formermethod is suitable for the preparation of a high-purity antibody, whilethe latter method is suited for high production. The antibody producedin this manner can be purified by the routine procedure such as saltingout, gel filtration, affinity chromatography or the like.

[0092] The antibody thus obtained is characterized in that it is capableof binding the protein of the invention. This characteristic can beexploited for the purification of the protein of the invention and theassay and identification of the protein by animmunological technique.The invention further provides such a novel antibody.

[0093] Based on the sequence information on the gene of the invention,which has been generated by the invention, the expression of the gene ofthe invention in the individual or in various tissues can be detected byutilizing a part or the whole of the nucleotide sequence of said gene.

[0094] In the invention, for the purpose of detecting the presence ofaTSC403 gene orhuman ING1L gene whose expression level is elevated in acancer tissue, one may prepare a biological sample, such as a blood orserum sample, optionally extract the DNA, and carry out an analysis tosee whether the sample contains a susceptible TSC403 gene or human ING1Lgene.

[0095] In accordance with the invention, for the purpose of detectingthe presence of a marker of malignancy in cells or tissues, progressionof malignancy to a prodromal disturbance, or prognosis, a biologicalsample of malignancy is prepared and analyzed for the presence of aTSC403 or human ING1L oncogene. By utilizing this technique, thepresence of such a marker of malignancy in cells or a tissue,progression of malignancy to a prodromal disturbance, orprognosis can bedetected. Therefore, the invention enables the diagnosis of a cancer,evaluation of the effect of a cancer therapy or prediction of theprognosis of a cancer.

[0096] The above detection can be carried out as follows. For example,based on the information on TSC403 gene or human ING1L gene as obtainedby using a sample from a tumor-bearing patient, a DNA fragment designedfor use in the screening for TSC403 gene or human ING1L gene and/or theamplification of the gene is first prepared. The DNA fragment mentionedabove includes the following.

[0097] (1) The fragment having the nature of a probe for plaquehybridization, colony hybridization, Southern blotting, Northernblotting, etc.

[0098] (2) The fragment having the nature of a probe for preparation ofthe entire or partial DNA fragment of TSC403 gene or human ING1L gene asamplified by PCR, that is a polymerase chain reaction for amplifying anucleotide sequence with a polymerase.

[0099] For the construction of such DNA fragments, a primer having thesame sequence as TSC403 gene or human ING1L gene is first prepared.Using this primer as a screening probe, it is reacted with a biologicalsample (nucleic acid sample) to confirm the presence of a gene havingthe TSC403 gene sequence or human ING1L gene sequence.

[0100] The above nucleic acid sample can be prepared by various methodsproviding for easy detection of the target sequence, such asdenaturation, restricted digestion, electrophoresis or dot blotting.

[0101] The method of said screening is preferably PCR from thestandpoint of sensitivity. This method is not particularly restrictedinasmuch as it employs a TSC403 gene fragment or a human ING1L genefragment as the primer and it may be any of the known protocols[Science, 230: 1350-1354 (1985)] and all PCR versions that are newlydeveloped or expected to be used in the future [Sakaki, Y. et al. (ed.),Jikken Egaku (Experimental Medicine), Supplement 8(9) (1990), Yodosha;Protein·Nucleic Acid·Enzyme; Special Supplement, Kyoritsu PublishingCo., 35(17) (1990)].

[0102] The DNA fragments for use as primers are chemically synthesizedoligo-DNAs. Those oligo-DNAs can be synthesized by using an automaticDNA synthesizer, e.g. Pharmacia LKB Gene Assembler Plus (Pharmacia). Thelength of the primer (sense primer or antisense primer) may for examplebe the equivalent of about 10-50 nucleotides, more preferably about15-30 nucleotides.

[0103] The probe for use in the above screening is usually a labeledprobe but may be an unlabeled probe. The screening may depend onspecific binding with a directly or indirectly labeled ligand. Themethod of labeling a probe or a ligand is known in the art and therelevant prior art includes nick translation, random priming, and kinasetreatment, among others. The substance which can be used as the labelincludes radioisotopes, biotin, fluorescent groups, chemiluminescentgroups, enzymes and antibodies which can be taken up by way of suchknown methods.

[0104] The above-mentioned detection can be performed in the routinemanner. For example, RNA amplification by RT-PCR [reversetranscribed-polymerase chain reaction; E. S. Kawasaki, et al.,Amplification of RNA. In PCR Protocol, A Guide to Methods andApplications, Academic Press, Inc., SanDiego, 21-27 (1991)], Northernblot analysis [Molecular Cloning, Cold Spring Harbor Lab. (1989)],determination on the cellular level, e.g. in situ RT-PCR [Nucl. AcidsRes., 21: 3159-3166 (1993)] and insitu hybridization, NASBA method[nucleic acid sequence-based amplification, Nature, 350: 91-92 (1991)],modifications of these techniques which are known in the art, andvarious other methods can invariably be used with success.

[0105] The method for assay according to the invention can be carriedout expediently by utilizing an assay reagent kit for detection ofTSC403 gene or human ING1L gene in samples. The invention furtherprovides an assay kit for detection of TSC403 gene or human ING1L genewhich contains said TSC403 gene fragment or human ING1L gene fragment.

[0106] It is important that this assay kit contain at least a DNAfragment hybridizing to a part or the whole of the nucleotide sequenceof SEQ ID NO:2 or 5 or a complementary nucleotide sequence thereto as anessential component. As the other components, the kit may contain alabeling agent and reagents necessary for PCR, such as Taq DNApolymerase, deoxynucleotide triphosphate, and primers, among others.

[0107] The labeling agent includes radioisotopes and chemical modifierssuch as fluorescent substances. These may be used as pre-conjugated tothe DNA fragment.

[0108] For convenience in practice of the assay, the assay kit of theinvention may contain a suitable reaction diluent, a standard antibody,a buffer, a washing buffer, a reaction stopper solution and so forth.

[0109] The invention further provides a method for cancer diagnosisutilizing the above assay method, a diagnostic reagent for use in saiddiagnosis, and a diagnostic kit.

[0110] By sequencing the TSC403 gene or human ING1L gene in a testsample as obtained by using the above assay method of the invention,either directly or indirectly by the conventional procedure, it ispossible to discover a novel TSC403 or human ING1L-related gene (mutantgene) which is highly homologous to the wild type TSC403 gene or wildtype human ING1L gene. Therefore, the invention further provides amethod of screening for a TSC403-related gene or human ING1L -relatedgene in a test sample which comprises performing said assay andsequencing the TSC403 gene or human ING1L gene in the sample.

[0111] Moreover, by utilizing the protein encoded by the TSC403 gene orhuman ING1L gene of SEQ ID NO:1 or 4, a protein having an amino acidsequence resulting from the deletion, substitution or addition of one ora plurality of amino acids from, in or to said sequence of SEQ ID NO:1or 4, or an antibody against such a fragment (polyclonal antibody ormonoclonal antibody; hereinafter referred to as “TSC403 antibody” or“human ING1L antibody”), said wild type TSC403 gene, wild type humanING1L gene, mutant TSC403 gene and mutant human ING1L gene can besuccessfully assayed.

[0112] The invention further provides a method for assay of such a wildtype TSC403 gene, a wild type human ING1L gene, a mutant TSC403 gene ora mutant human ING1L gene.

[0113] According to this assay methodology, the severity of disturbancein a neoplastic state or the malignancy of a neoplasm can be detectedfrom a change in the wild type TSC403 gene or wild type human ING1Lgene. The change mentioned above can be determined or detected by thesequencing of the TSC403 gene or human ING1L gene by any of theabove-mentioned conventional sequencing techniques, and more preferablyby the assay method using said TSC403 antibody or human ING1L antibody.In this manner, the presence of an anomaly (mutation) in the TSC403protein or human ING1L protein in a test sample or the presence orabsence of the TSC403 protein or human ING1L protein can be detected.

[0114] In the assay procedure of the invention which utilizes ananti-TSC403 antibody or anti-human ING1L antibody, the antibody can beused to immunoprecipitate the TSC403 protein or human ING1L protein froma solution containing a biological material obtained from a humansubject, such as blood or serum, or caused to react with TSC403 proteinor human ING1L protein on the polyacrylamide gel Western blot or on theimmunoblot.

[0115] Furthermore, by utilizing the anti-TSC403 antibody or anti-humanING1L antibody in an immunohistochemical assay procedure, the TSC403protein or human ING1L protein in a paraffin section or a frozen tissuespecimen can be detected. The production technology and purificationprocedure which can be used for said anti-TSC403 antibody or anti-humanING1L antibody are well known in the art. Such known techniques can beutilized for the production and purification of said antibody.

[0116] The more preferred protocol relevant to the detection of a wildtype TSC403 or human ING1L, or a mutant thereof, includes a sandwichassay using a monoclonal antibody and/or a polyclonal antibody. Amongother preferred detection techniques are enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA)and immunoenzymometric assay (IEMA).

[0117] The invention further provides a TSC403 protein receptor or humanING1L protein receptor existing in a cell membrane fraction or a cellsurface and having binding activity for TSC403 protein or human ING1Lprotein. This TSC403 protein receptor or human ING1L protein receptorcan be produced and obtained, for example by adding a labeled TSC403protein or human ING1L protein to a cell membrane fraction containingthe receptor or a biological sample containing the same, extracting,isolating and purifying the resulting receptor-protein conjugate (TSC403protein-binding reaction product or human ING1L protein-binding reactionproduct) and identifying the amino acid sequence of the isolatedproduct. The preparation and sequencing of the TSC403 protein or humanING1L protein receptor can be easily made by those skilled in the artaccording to the established procedures.

[0118] The TSC403 protein receptor or human ING1L protein receptoraccording to the invention, or fragments thereof, can be applied to thescreening for various drugs. By such technology, compounds capable ofreacting with said receptor (low molecular compounds, high molecularcompounds, proteins, protein fragments, antigens, antibodies, etc.) canbe screened out. The receptor, or a fragment thereof, which is to beused in such screening can be put to use as immobilized on a suitablesolid matrix or in the form of a free substance in a solutiontransported to the cell surface.

[0119] An example of the above pharmacoscreening is the screening inwhich procaryotic or eucaryotic host cells transformed stably with arecombinant protein expressing the TSC403 protein or human ING1Lprotein, or a fragment thereof, are used in, preferably, a competitivebinding assay. As an alternative, said host cells, whether in the freeform or as immobilized, are used in the standard binding assay. Moreparticularly, the above pharmacoscreening may comprise reacting theTSC403 protein receptor or human ING1L protein receptor, or a fragmentthereof, with the TSC403 protein or human ING1L protein, or a fragmentthereof, in the presence of a candidate drug, to cause formation of acomplex and detecting the degree of inhibition of the complex formationby the above candidate drug.

[0120] Thus, in accordance with the invention, there can be provided amethod for pharmacoscreening which comprises contacting a candidate drugwith the TSC403 protein receptor or human ING1L protein receptor, or afragment thereof and, then, detecting the presence of the resultingcomplex or the presence of a complex of the TSC403 protein receptor orhuman ING1L protein receptor, or a fragment thereof, and the ligand by aper se known technique.

[0121] Furthermore, by assaying TSC403 protein receptor activity orhuman ING1L protein receptor activity, it is possible to evaluatewhether a candidate drug is capable of antagonizing the TSC403 proteinreceptor or human ING1L protein receptor and accordingly inhibitingTSC403 protein activity or human ING1L protein activity, for examplemitosis-promoting activity.

[0122] In such a competitive binding assay, the TSC403 protein receptoror human ING1L protein receptor, or a fragment thereof, is labeled. Whenthe free TSC403 protein receptor, human ING1L protein receptor orfragment thereof is separated from the corresponding complex and thelabeling amount of the free (non-complex-forming) substance is measured,the measured value serves as a yardstick of the binding of the candidatedrug to the TSC403 protein receptor or human ING1L protein receptor.Furthermore, said measured value serves also as a measure of inhibitionof the binding of the TSC403 protein receptor or human ING1L proteinreceptor to the TSC403 protein or human ING1L protein. By analyzing asmall peptide (pseudopeptide) of TSC403 protein or human ING1L proteinin this manner, the candidate drug can be assayed as a substance havingTSC403 protein receptor antagonizing activity or human ING1L proteinreceptor antagonizing activity.

[0123] Another protocol for pharmacoscreening in accordance with theinvention is that of screening for a compound having an adequate bindingaffinity for the TSC403 protein receptor or human ING1L proteinreceptor. Briefly, this procedure comprises synthesizing a large numberof different test peptide compounds on a solid support such as thesurface of a plastic pin or other material, reacting the test compoundswith the TSC403 protein receptor or human ING1L protein receptor and,after washing, detecting the binding reaction products by a known method[e.g. PCT patent publication No. WO 84-03564].

[0124] The purified TSC403 protein receptor or human ING1L proteinreceptor can be directly coated on the plate to be used in saidpharmacoscreening procedure. Moreover, the antibody may be captured witha non-neutralizing antibody against the polypeptide and the TSC403protein receptor or human ING1L protein receptor be immobilized on asolid phase.

[0125] The invention is further directed to the use of a competitivepharmacoscreening assay. For the binding to the TSC403 protein receptoror human ING1L protein receptor, or a fragment thereof, a neutralizingantibody capable of specific binding to the TSC403 protein receptor orhuman ING1L protein receptor is caused to compete with the candidatecompound. By such a competitive reaction with the neutralizing antibody,the presence of any peptide having one or more antigenic determinants ofthe TSC403 protein receptor or human ING1L protein receptor can bedetected.

[0126] Furthermore, in connection with pharmacoscreening, a stillanother method comprises the use of a host eucaryotic cell line or cellscontaining a nonfunctional TSC403 gene or nonfunctional human ING1Lgene. The host cell line or cells are caused to multiply in the presenceof a candidate drug for a predetermined time and the velocity of growthof the host cells is determined to see whether, for example, thecandidate drug is capable of inhibiting growth of the cells. The meansfor measuring said velocity of growth includes a method of determiningthe biological activity of the TSC403 protein receptor or human ING1Lprotein receptor.

[0127] Furthermore, in accordance with the invention, for thedevelopment of a more active or stable derivative of TSC403 protein orhuman ING1L protein or of a drug which will enhance or interfere withthe function of TSC403 protein or human ING1L protein in vivo, it ispossible to construct bioactive proteins, or their structural analogs,with which said protein would interact, such as a TSC403 protein orhuman ING1L protein receptor agonist, a TSC403 protein or human ING1Lprotein receptor antagonist, or a TSC403 protein or human ING1L proteininhibitor, for instance. The structural analogs mentioned above can becharacterized by analysis of the three-dimensional structure of acomplex between TSC403 protein or human ING1L protein and a third-partyprotein by X-ray crystallography or computer modeling or a combinationof such techniques. The structural information on such structuralanalogs can also be obtained by protein modeling based on the structuresof homologous proteins.

[0128] As a method for providing said more active or stable TSC403protein derivative or human ING1L protein derivative, there can bementioned an alanine scan technique. This technique comprisessubstituting an alanine residue for each of certain amino acid residuesof said protein and determining the effect of substitution on theactivity of the resulting protein. In other words, this technique issuch that by said substitution for amino acid residues of the proteinand analysis, the domain of significance to the activity or stability ofthe protein is determined. This technique enables design of a moreactive or stable TSC403 protein derivative or human ING1L proteinderivative.

[0129] Furthermore, it is now possible to isolate a target-specificantibody selected by a functional assay and analyze its crystalstructure. As a rule, the pharmacore providing a basis for subsequentdrug design can be obtained by this approach. Through the generation ofan anti-idiotype antibody for a functional pharmacoactive antibody, apeptide can be identified or isolated from a chemically or biologicallyconstructed peptide bank. Hence, the selected peptide is also expectedto serve as a pharmacore.

[0130] Thus, it is now possible to design and develop drugs havingTSC403 protein or human ING1L protein inhibitor, agonist or antagonistactivity for TSC403 protein or human ING1L protein having improvedactivity, stability and other characteristics.

[0131] It is also possible to prepare a sufficient amount of TSC403protein or human ING1L protein by using a cloned TSC403 gene or clonedhuman ING1L gene and carry out X-ray crystallographic and otheranalytical investigations. Furthermore, as the result of provision ofthe TSC403 protein or human ING1L protein of SEQ ID NO:1 or 4 accordingto the invention, it is now possible to provide a computer modelingprogram or technique as a substitute for X-ray crystallography or as anadjunct thereto.

[0132] The invention enables construction of a TSC403 gene-bearingknockout mouse (mutant mouse) or human ING1L gene-bearing knockout mouse(mutant mouse). By this approach, it can be ascertained which region ofthe nucleotide sequence of the TSC403 gene or human ING1L geneinfluences said divergent activities of TSC403 protein or human ING1Lprotein in vivo, that is to say what functions TSC403 gene products,human ING1L gene products, modified-TSC403 gene products ormodified-human ING1L gene products would have in vivo.

[0133] This is a technique for modifying the genetic information of anorganism intentionally by utilizing a homologous recombination of genes,and a protocol using mouse embryonic stem cells (ES cells) is known[Capeccchi. M. R., Science, 244, 1288-1292 (1989)].

[0134] The above construction of mutant mice belongs to the expertise ofthose killed in the art and the wild type TSC403 gene, wild type humanING1L gene, mutant TSC403 gene or mutant human ING1L gene according tothe invention can be subjected to this modification [Tetsuo Noda (ed.):Jikken Igaku (Experimental Medicine), Supplement, 14(20) (1996),Yodosha] to thereby construct said respective mutant mice in anexpedient manner. By utilizing this technique, it is possible to designand develop drugs having TSC403 protein or human ING1L proteininhibitor, agonist or antagonist activity for the protein havingimproved TSC403 protein activity or stability or improved human ING1Lprotein activity or stability.

[0135] Thus, the invention further provides a specific primer fordetection of the TSC403 gene or human ING1L gene of the invention and/ora DNA fragment for use as the specific primer, a method for cancerdiagnosis which utilizes them, and a diagnostic kit therefor.

[0136] For example, the TSC403 gene probe according to the invention canbe produced and acquired by the standard PCR technique using two kindsof primers (sense primer and antisense primer) which are specific to theTSC403 gene of the invention. With the probe thus constructed,expression of the genes of the invention in various neoplastic and othertissues can be detected.

BRIEF DESCRIPTION OF DRAWINGS

[0137]FIG. 1 is a photograph substituting for a drawing which shows adistribution of the TSC403 gene of the invention in human tissues asfound by the Northern blot analysis in accordance with Example 1-(2).

[0138]FIG. 2 is a photograph substituting for a drawing which shows theresults of the RT-PCR analysis of various normal tissues and cancertissues in accordance with Example 1-(4).

[0139]FIG. 3 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example 1-(5).

[0140]FIG. 4 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example 1-(5).

[0141]FIG. 5 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example 1-(5).

[0142]FIG. 6 is a photograph substituting for a drawing which showscontrol cells in the focus forming test in accordance with Example1-(6).

[0143]FIG. 7 is a photograph substituting for a drawing which showstransformant cells as transformed with the TSC403 gene of the inventionin the focus forming test in accordance with Example 1-(6).

[0144]FIG. 8 shows the result of a homology study between the predictedamino acid sequence of the protein encoded by the human ING1L gene ofthe invention and that of p33^(ING1) [GenBank A. C. No. AF044076].

[0145]FIG. 9 is a photograph substituting for a drawing which shows theresults of a Northern blot analysis of 16 human adult organ-derivedcells in accordance with Example 2-(2).

[0146]FIG. 10 is a photograph substituting for a drawing which shows theresults of a Northern blot analysis of the colorectal cancer patienttissue in accordance with Example 2-(2).

BEST MODE FOR CARRYING OUT THE INVENTION

[0147] The following examples are intended to illustrate the inventionin further detail.

EXAMPLE 1

[0148] TSC403 Gene

[0149] (1-1) Procedure for Imaging by [γ-³³P]ATP Labeling

[0150] For confirmation of the human gene expressed by a tissue-specifictechnique, the [γ-³³P]ATP-labeled imaging method was used. This methodwas essentially carried out according to the protocol of Liang [LiangP., et al., Science, 257, 967-971 (1992)].

[0151] Thus, the poly A RNA (0.2 μg) isolated from each of 13 humantissues (adult brain, fetal brain, lung, liver, stomach, pancreas,spleen, mammary gland, prostate, placenta, testis, kidney and heart;Clontech) was mixed with 25 pmol of 3′-anchored oligo-dT primer G(T) 15MA (M stands for a mixture of G, A and C) in 8 μl of diethylpyrocarbonate-treated water and the mixture was heated at 65° C. for 5minutes. To this solution, 4 μl of 5×first strand buffer (BRL), 2 μl of0.1 M DTT (BRL), 1 μl of 250 mM dNTPs (BRL), 1 μl of ribonucleaseinhibitor (40 units; Toyobo) and 1 μl of Superscript II reversetranscriptase (200 units; BRL) were added. The final volume of eachreaction mixture was 20 μl. Each solution was incubated at 37° C. for 1hour and diluted 2.5-fold by adding 30 μl of distilled water and thedilution was stored at −20° C. until used.

[0152] The cDNA was amplified by PCR in the presence of[γ-³³P]ATP-labeled (Pharmacia) 3′-anchored primer. This PCRamplification of cDNA was carried out as follows.

[0153] Thus, 20 μl of each PCR mixture contained 2 μl of RT reactionmixture, 2 μl of 10×PCR buffer (Takara), 4 μl of 2.5 mM dNTPs, 0.25 μlof ExTaq DNA polymerase (5 U/ml; Takara), 25 pmol of 3′-anchoredoligo-dT primer labeled with [α-³³P]ATP, and 25 pmol of 5′-primer (No.20, a 10-mer deoxyoligonucleotide primer having a randomized sequence ofthe sequence SEQ ID NO:7). The PCR was carried out under the followingconditions: one cycle of 95° C. for 3 min., 40° C. for 5 min. and 72° C.for 5 min.; 40 cycles of 95° C. for 0.5 min., 40° C. for 2 min. and 72°C. for 1 min.; and annealing at 72° C. for 5 min.

[0154] The PCR sample was extracted with ethanol, resuspended informamide-sequencing dye, and reacted on the 6% acrylamide-7.5 M ureasequencing gel. The gel was dried without fixing and autoradiography wasperformed overnight.

[0155] (1-2) Sub-cloning of the Amplified cDNA Fragment

[0156] A 3 MM filter paper, on which the dry gel was placed in advance,was marked in a radioactive ink and the autoradiogram was set inregistry with the marking. The gel containing the objective cDNA bandwas cut out together with the 3 MM filter paper and stirred in 300 μl ofdH₂O for 1 hour. After removal of the polyacrylamide gel and filterpaper, the cDNA was recovered by ethanol precipitation in the presenceof 1 μl of 10 mg/ml glycogen and 0.3 M NaOAc as the carrier andredissolved in 10 μl of dH₂O. For reamplification, 5 μl of this solutionwas used. The PCR conditions and primers used were the same as thoseused for the first PCR. The reamplification product of a suitable sizewas recovered as the first PCR product and this PCR product was clonedin the Hinc II site of pUC118 vector (Takara). The nucleic acid sequencewas determined by using ABI377 Automatic Sequencer (Applied BioSystems).

[0157] By comparing the various image patterns obtained with the mRNAsisolated from 13 kinds of human tissues, one PCR product expressedspecifically in the lung could be identified. This product was namedTSC403DD.

[0158] This product consisted of 252 nucleotides,. Comparison of thisnucleotide data with the DNA sequences in the GenBank/EMBL Databaseusing FASTA Program [Person, W. R., et al., Proc. Natl. Acad. Sci., USA,85, 2444-2448 (1988)] revealed that this PCR product has no homology toany known DNA sequence.

[0159] (1-3) cDNA Screening

[0160] A human normal lung cDNA library was constructed usingoligo(dT)+random hexamer-primed human normal lung cDNA and Uni-ZAP™ XR(Stratagene). All the 1×10⁶ clones were isolated by the above-describedprocedure and a screening was carried out using the [α-³²P]-dCTP-labeledcDNA fragment. Positive clones were selected and the cloned sites ofcDNA were excised in vivo in pBluescript II SK(−).

[0161] As a result, about 100 plaques were identified for said TSC403DD.Based on this result, the transcription amount in the total RNApopulation was calculated to be about 0.01%.

[0162] The assembled cDNA sequence (TSC403) homologous to TSC403DDcomprised 3198 nucleotides inclusive of a 1248-nucleotide open readingframe coding for a 416-amino acid residue protein having a calculatedmolecular mass of 44316 Da.

[0163] From the primary sequence, this gene product (TSC403 protein) wasfound to be a protein containing a transmembrane domain.

[0164] Its chromosomal locus was found to be 3q27 where chromosomalaberrations are noted in various kinds of cancers.

[0165] Furthermore, the gene TSC403 of the invention has about 30%homology to human lamp-1 and lamp-2.

[0166] (2) Expression in Tissues

[0167] To delineate the expression profile of TSC403 in tissues,Northern blotting was performed using various human tissues.

[0168] In the Northern blot analysis, human MTN (Multiple-TissueNorthern) blot I and II (Clontech) were used. The cDNA fragment wasprepared by using a primer set of T3 and T7 promoter sequences andlabeled with [α-³²P]-dCTP by PCR. The membrane containing theamplification product was subjected to the prehybridization (under theconditions of the product protocol) and further to hybridizationaccording to the product protocol.

[0169] After hybridization, the membrane was washed and exposed forautoradiography at −80° C. for 24 hours. The results are shown in FIG.1.

[0170] The human tissues used and represented on the drawing are heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon and peripheralblood leukocyte; P.B.L.).

[0171] As can be seen from the drawing, a transcript homologous toTSC403 was specifically detected in the lung.

[0172] (3) FISH

[0173] FISH for chromosome mapping was carried out by the known method[Takahashi E., et al., Hum. Genet., 86, 14-16 (1990)] using 0.5 μg ofeach cosmid DNA as a probe. FISH was caught by Provia 100 film (Fuji;ISO 100) or with a CCD camera system (Applied Imaging, Cyto Vision).

[0174] As a result, the 100 signals obtained with the cells in thetypical R-band (pro) metaphase were localized in the 3q27 band.Therefore, the chromosomal locus of TSC403 could be identified to be3q27.

[0175] (4) Expression in Cancer Cell Lines and Cancer Tissues asAnalyzed by RT-PCR

[0176] To investigate whether the expression of the TSC403 gene would bemutated in human cancer cell lines and cancer tissues, four cell lines[Aspc1 (metastatic adenocarcinoma, J. Natl. Cancer Inst., 67, 563-569(1981)), Bxpc3 (adenocarcinoma, undifferentiated; Cancer Invest., 4,15-23 (1986)), MiaPaca2 (adenocarcinoma, Int. J. Cancer, 19, 128-135(1977)) and PANC1 (epithelioid, carcinoma of pancreatic duct, Int. J.Cancer, 15, 741-747 (1975)) and 9 cancer tissues (donated by Dr.Nakamura, The Institute of Medical Science, the University of Tokyo)]were subjected to RT-PCR analysis.

[0177] Thus, 10 μl of the whole RNA isolated from each cell line orcancer tissue by using ISOGEN (Wako Chemical Ind.) was treated with 10units of RNase-free DNase I (Boehringer Mannheim) for 15 minutes,extracted twice with phenol-chloroform, and precipitated from ethanol.The single-stranded cDNA was synthesized by means of Superscript I™RNase H-reverse transcriptase (Life Technology) using oligo-(dT) andrandom primers. A 2 μl-portion of each product was used for PCRamplification.

[0178] The primers P1 and P2 having the nucleotide sequences depicted inSEQ ID NO:8 and SEQ ID NO:9 were used for 25-cycle PCR amplification.

[0179] The PCR was carried out in 20 μl of a solution of DNA 25 ng,primers 10 μM each, dNTP 2.5 mM and Extaq DNA polymerase (Takara) 0.25U. The PCR product was dissolved in ethidium bromide-stained 1.5%agarose gel.

[0180] The results of the RT-PCR analysis of 4 cell lines (lane1=AsPc-1, Lane 2=BxPc-3, Lane 3=MIApaca, lane 4=PANC-1), normalpancreatic tissue (Normal pancreas, lanes 1 and 2), pancreatic cancertissue (Pancreatic cancer, lanes 1-11) and normal lung tissue (Normallung) by the above procedure are shown in FIG. 2.

[0181] It is clear from FIG. 2 that the expression of TSC403 was notfound in the normal pancreatic tissue (Normal pancreas, lanes 1 and 2)but found exclusively in the pancreatic cancer tissue (Pancreaticcancer, lanes 1-11 and Cell lines, lanes 1-4).

[0182] Incidentally, the 4 cell lines used above have been depositedwith ATCC and their accession numbers are as follows.

[0183] Aspc-1; CRL-1682

[0184] BxPc-3; CRL-1687

[0185] MIApaca; CRL-1420

[0186] PANC-1; CRL-1469

[0187] (5) Expression of the TSC403 Gene in Various Cancers (NorthernBlot Analysis)

[0188] Expression of the TSC403 gene was studied by hybridizing theTSC403 gene to the blot (Invitrogen carrying the following variouscancer tissue and normal tissue mRNA samples (tumor Northern blotanalysis). All the cancer tissues and normal tissues used were purchasedfrom Invitrogen.

[0189] The results are shown in FIG. 3, FIG. 4 and FIG. 5. The cancertissues and normal tissues represented on each drawing are as follows.

[0190]FIG. 3:

[0191] Brain tumor, brain normal, kidney tumor, kidney normal, livertumor, liver normal, lung tumor, lung normal, breast tumor, normalbreast, uterine tumor, normal uterine, fallopian tube tumor, normalfallopian tube, ovarian tumor, normal ovary.

[0192]FIG. 4:

[0193] Esophagus tumor, normal esophagus, stomach tumor, normal stomach,colon tumor, normal colon, rectum tumor, normal rectum, thyroid tumor,normal thyroid, adrenal tumor, normal adrenal, parotid tumor, normalparotid, lymphoma, normal lymph node.

[0194]FIG. 5:

[0195] Kidney tumor, normal kidney, ureter tumor, normal ureter, bladdertumor, normal bladder, stomach tumor, normal stomach, ovarian tumor (4cases), ovarian normal (4 cases).

[0196] From the above drawings, significant expression of TSC403 can befound in the normal lung. In addition, expression of the TSC403 gene wasobserved in breast tumor (FIG. 3), fallopian tube tumor (FIG. 3),esophagus tumor (FIG. 4), colon tumor (FIG. 4), rectum tumor (FIG. 4),thyroid tumor (FIG. 4), parotid tumor (FIG. 4), ureter tumor (FIG. 5),ovarian tumor (FIG. 5, 2 out of 4 cases).

[0197] (6) Focus Forming Assay by Expression of the TSC403 Gene

[0198] The full-length open reading frame of the TSC403 gene was ligatedto the BamHI-XhoI site of the pcDNA 3.1/His (Invitrogen) vector toobtain a TSC403 gene expression vector.

[0199] Then, using the expression vector obtained above, a focus formingassay using NIH3T3 cells was carried out according to the methoddescribed in the literature [Shin, C., Shilo, B., Goldfarb, M. P., etal., Proc. Natl. Acad. Sci., USA., 76, 5714-5718 (1979)] to see whetherthe TSC403 gene has a tumorigenic effect on cells.

[0200] The results are shown in FIG. 6 (control cells not transformedwith TSC403 gene) and FIG. 7 (cells transformed with TSC403 gene).

[0201] As is apparent from comparison of the two figures, a definitefocus was formed, as shown in FIG. 7, when the TSC403 gene wasforce-expressed in cells by introduction of the gene. It is, thus,apparent that the TSC403 gene, when over-expressed by force, causes aloss of sensitivity to the contact inhibition phenomenon which is one ofmalignant transformations of cells, thus being deeply involved in thetumorigenesis of cells.

EXAMPLE 2

[0202] Human ING1L Gene

[0203] (1) Cloning and DNA Sequencing of the Human ING1L Gene

[0204] By the sequencing of cDNA clones arbitrarily selected from ahuman fetal brain cDNA library and database search, one clone(GEN-146F11) harboring a cDNA coding for an amino acid sequence havinghigh homology to p33^(ING1), a protein considered to be atumor-suppressive protein, was isolated by the following procedure.

[0205] Thus, the mRNA extracted from human fetal brain was purchasedfrom Clontech and used as the starting material. From the mRNA, the cDNAwas synthesized and cloned into Vector λ ZAPII (Stratagene) to constructa cDNA library (Otsuka GEN Research Institute, Otsuka Pharmaceutical).By the in vivo excision method [Short, J. M. et al., Nucleic Acids Res.,16, 7583-7600 (1988)], colonies of Escherichia coli harboring the humangene were caused to form on agar medium and picked up at random and E.coli clones harboring the human gene were registered on a 96-wellmicrotiter plate. The registered clones were stored at −80° C.

[0206] Then, each registered clone was cultured in 1.5 ml of LB mediumfor 24 hours, and using a plasmid automatic extractor PI-100 (Kurabo),the DNA was extracted and purified. The contaminated E. coli RNA wasdecomposed by RNase treatment and removed. Finally, the DNA wasdissolved to 30 μl and a 2 μl portion was used for a rough estimation ofDNA size and amount by the minigel method. Another 7 μl portion was usedfor sequencing reaction and the remaining 21 μl portion was stored asplasmid DNA at 4° C.

[0207] Then, a Sanger's dideoxy terminator [Sanger, F., et al., Proc.Natl. Acad. Sci., U.S.A., 74, 5463-5467 (1977)] using T3, T7 or asynthetic oligonucleotide primer or a cycle sequencing method which isthe dideoxy terminator method combined with the PCR method [Carothers,A. M., et al., Bio. Techniques, 7, 494-499 (1989)] was carried out.These are the methods for chain extension reaction with terminationspecific to 4 kinds of bases using a small amount of plasmid DNA (ca0.1-0.5 g) as a template.

[0208] Using an FITC (fluorescein isothiocyanate)-labeled primer as thesequence primer, about 25 cycles of reaction using Taq polymerase werecarried out. Of the fluorescence-labeled DNA fragment, the sequence ofabout 400 nucleotides from the 5′-end of the cDNA was determined withthe automatic DNA sequencer ALF™ DNA Sequencer (Pharmacia).

[0209] The 3′-nontranslated region is high in heterogeneity among genesand suited for differentiation of individual genes. Therefore,sequencing of the 3′-end region was also performed in some cases.

[0210] The huge nucleotide sequence information generated with the DNAsequencer was transmitted to the 64-bit computer DEC3400 forcomputerized homology analysis. This homology analysis was carried outby a database (GenBank, EMBL) search according to UWGCG's FASTA Program[Pearson, W. R. and Lipman, D. J., Proc. Natl. Acad. Sci., USA., 85,2444-2448 (1988)].

[0211] The above method of analysis for a human fetal brain cDNA libraryis described in detail by Fujiwara et al. [Fujiwara, T., et al., DNARes., 2, 107-111 (1991)].

[0212] About 5040 ESTs (expressed sequence tags: partial DNA sequencesof expressed gene fragment) randomly selected from the human fetal braincDNA library constructed as above were then sequenced.

[0213] The clone named GEN-146F11 in the GenBank/EMBL sequence searchaccording to the FASTA Program was found to harbor a gene coding for anamino acid sequence having high homology to p33^(ING1) [GenBank A. C.No. AF001954].

[0214] To clarify the full-length sequence in said GEN-146F11 clone, aDNA sequencing reaction using T7DNA polymerase and a synthetic primerwas carried out. In addition, using a double-stranded DNA inserted intoa vector (pBluescript vector; Stratagene) as a template and a syntheticoligonucleotide as a primer, the nucleotide sequence of the cDNAinclusive of the whole coding region was determined by Sanger's dideoxychain termination method, and the sequence was compared with the DNAsequences of several other related genes.

[0215] SEQ ID NO:6 shows the nucleic acid sequence of GEN-146F11 clone(cDNA); SEQ ID NO:5 shows the nucleic acid sequence of the coding regionof said clone; and SEQ ID NO:4 shows the deduced amino acid sequenceencoded by said nucleic acid sequence.

[0216] In the above nucleotide sequences, the initiation signal sequencewas found in the position 92-94 and suspected to be the translationstart codon. The predicted stop codon was found in the position 932-934.

[0217] The cDNA has a length of 1078 nucleotides and contained an openreading frame of 840 base pairs that coded for a predicted 280-aminoacid residue protein.

[0218] By the homology search using FASTA Program, this gene was foundto code an amino acid sequence having high homology to p33^(ING1)[GenBank A. C. No. AF044076]. The homology of the nucleotide sequencewas 60.0%.

[0219] On the amino acid sequence level, the homology between thededuced amino acid sequence of the protein encoded by the gene of theinvention and the sequence of p33^(ING1) [GenBank A. C. No. AF044076]was investigated. The result is shown in FIG. 8.

[0220]FIG. 8 shows the amino acid sequence depicted in single letters;the top row represents the sequence of the human ING1L protein encodedby the gene of the invention (indicated as hING1L) and the bottom rowrepresents the sequence of p33^(ING1) [Garkavetsev, et al., Nature.Genet., 14, 415-420 (1996); Garkavetsev, et al., Mol. Cell. Biol., 17,2014-2019 (1997), GenBank A. C. No. AF001954; however this sequence hasbeen revised subsequently and the sequence as corrected is shown inGenBank A. C. No. AF044076; indicated as p33^(ING1) on the drawing].

[0221] Furthermore, on the same drawing, the solid area (black frame)indicates the identical amino acid residues and the shaded area (shadedframe) indicates analogous amino acid residues. The symbol ---- in thehING1L row stands for a gap.

[0222] It can be seen from the drawing that the amino acid sequenceencoded by the gene of the invention has 58.9% (as calculated based onthe sequence as corrected of p33^(ING1)) homology to the amino acidsequence of p33^(ING1).

[0223] (2) Northern Blot Analysis

[0224] The expression of human ING1L mRNA in normal human tissues wasevaluated by Northern blotting using a human cDNA clone labeled by therandom oligonucleotide priming method as a probe.

[0225] The Northern blot analysis was performed using a human MTN blot(Human Multiple Tissue Northern Blot; Clontech) according to the productprotocol.

[0226] Thus, the full-length sequence of said clone GEN-146F11 wasPCR-amplified and the PCR product was labeled with [³²P]-dCTP (RandomPrimed DNA Labeling Kit, Boehringer Mannheim) for use as a probe.

[0227] The blot was subjected to 4-hour prehybridization and, then, tohybridization in a solution of 50% formamide/5×SSC/10×Decherdsolution/2% SDS solution (containing 100 μg/ml of denatured salmon spermDNA) at 42° C. overnight. After two washings with 2×SSC/0.1% SDS at roomtemperature, 2 washings were carried out with 0.2×SSC/0.1% SDS at 65° C.for 15 minutes. The filter was exposed against X-ray film (Kodak) at−70° C.

[0228] The results of 18-hour exposure are shown in FIG. 9.

[0229] As can be seen in FIG. 9, the expression was found in all the 16human adult organ-derived tissues tested (heart, brain, placenta, lung,liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,testis, uterus, small intestine, colon, peripheral blood and leukocyte;the same nomenclature applies to the legends on the drawing), and twotranscripts of 1.5 kb and 1.3 kb were detected.

[0230] Furthermore, for the several tumor tissues of colorectal cancer,cancer of esophagus, cancer of uterine tube and stomach cancer, too, asimilar Northern blot analysis was carried out using a human TP blot(Human Tumor Panel Blot; Invitrogen) in accordance with the productprotocol.

[0231] The results in colorectal cancer patient tissues are shown inFIG. 10.

[0232] On the drawing, T represents the colorectal tumor tissue(indicated as T:Tumor on the drawing) and N represents the normalcolorectal tissue (indicated as N:Normal on the drawing). One set of Tand N is the tissue derived from one patient and the drawing shows theresults for tissues derived from 4 patients.

[0233] It is clear from the drawing that in each individual patient, thelevel of expression of the human ING1L gene is elevated in the canceroustissue as compared with the normal tissue.

[0234] Based on the above findings, it is thought that the human ING1Lgene of the invention is useful for cancer research and therapy,particularly for application to cancer diagnosis, and that if anyantagonistic inhibitor of expression products of the human ING1L gene bedeveloped in the future, it should find application as an anticanceragent.

[0235] (3) Chromosome Mapping by FISH and Radiation HybridizingTechniques

[0236] FISH for chromosome alignment was carried out by the knownprocedure [Takahashi, E. et al., Hum. Genet., 86, 14-16 (1990)] using0.5 μg of each cosmid DNA as a probe. FISH was caught by Provia 100 film(Fuji, ISO 100) or with a CCD Camera System (Applied Imaging, CytoVision).

[0237] As a result, signals of 100 typical R-band (pro) metaphase cellsindicated that the locus of the human ING1L gene on a chromosome was4q35.1.

Industrial Applicability

[0238] In accordance with the invention, there is provided not only anovel lung-specific gene TSC403 but also a protein encoded thereby.Through utilization thereof, there is provided a technology by whichmore light may be cast on cancers, e.g. lung cancer and pancreaticcancer, and the process of oncogenesis and which finds application inthe diagnosis, prophylaxis and therapy thereof.

[0239] Further provided in accordance with the invention is a novelhuman ING1L gene which enables detection of the expression of the genein various tissues, production of a human ING1L protein, which is theexpression product of the gene, by genetic engineering technology, andconstruction of a specific antibody against said protein. These, inturn, enable research into the cell cycle, inhibition of growth oractivation of various cells, study of metabolic agingandapoptosis ofcells, and exploration, treatment or diagnosis of related diseases suchas cancers, as mentioned hereinbefore. In addition, the inventionenables the development of, or screening for, antagonistic inhibitors ofsaid human ING1L protein, namely cell growth suppressants and anticancerdrugs.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1<211> LENGTH: 416 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: Human normal lung cDNA library <400>SEQUENCE: 1 Met Pro Arg Gln Leu Ser Ala Ala Ala Ala Leu Phe Ala Ser LeuAla 1 5 10 15 Val Ile Leu His Asp Gly Ser Gln Met Arg Ala Lys Ala PhePro Glu 20 25 30 Thr Arg Asp Tyr Ser Gln Pro Thr Ala Ala Ala Thr Val GlnAsp Ile 35 40 45 Lys Lys Pro Val Gln Gln Pro Ala Lys Gln Ala Pro His GlnThr Leu 50 55 60 Ala Ala Arg Phe Met Asp Gly His Ile Thr Phe Gln Thr AlaAla Thr 65 70 75 80 Val Lys Ile Pro Thr Thr Thr Pro Ala Thr Thr Lys AsnThr Ala Thr 85 90 95 Thr Ser Pro Ile Thr Tyr Thr Leu Val Thr Thr Gln AlaThr Pro Asn 100 105 110 Asn Ser His Thr Ala Pro Pro Val Thr Glu Val ThrVal Gly Pro Ser 115 120 125 Leu Ala Pro Tyr Ser Leu Pro Pro Thr Ile ThrPro Pro Ala His Thr 130 135 140 Ala Gly Thr Ser Ser Ser Thr Val Ser HisThr Thr Gly Asn Thr Thr 145 150 155 160 Gln Pro Ser Asn Gln Thr Thr LeuPro Ala Thr Leu Ser Ile Ala Leu 165 170 175 His Lys Ser Thr Thr Gly GlnLys Pro Asp Gln Pro Thr His Ala Pro 180 185 190 Gly Thr Thr Ala Ala AlaHis Asn Thr Thr Arg Thr Ala Ala Pro Ala 195 200 205 Ser Thr Val Pro GlyPro Thr Leu Ala Pro Gln Pro Ser Ser Val Lys 210 215 220 Thr Gly Ile TyrGln Val Leu Asn Gly Ser Arg Leu Cys Ile Lys Ala 225 230 235 240 Glu MetGly Ile Gln Leu Ile Val Gln Asp Lys Glu Ser Val Phe Ser 245 250 255 ProArg Arg Tyr Phe Asn Ile Asp Pro Asn Ala Thr Gln Ala Ser Gly 260 265 270Asn Cys Gly Thr Arg Lys Ser Asn Leu Leu Leu Asn Phe Gln Gly Gly 275 280285 Phe Val Asn Leu Thr Phe Thr Lys Asp Glu Glu Ser Tyr Tyr Ile Ser 290295 300 Glu Val Gly Ala Tyr Leu Thr Val Ser Asp Pro Glu Thr Val Tyr Gln305 310 315 320 Gly Ile Lys His Ala Val Val Met Phe Gln Thr Ala Val GlyHis Ser 325 330 335 Phe Lys Cys Val Ser Glu Gln Ser Leu Gln Leu Ser AlaHis Leu Gln 340 345 350 Val Lys Thr Thr Asp Val Gln Leu Gln Ala Phe AspPhe Glu Asp Asp 355 360 365 His Phe Gly Asn Val Asp Glu Cys Ser Ser AspTyr Thr Ile Val Leu 370 375 380 Pro Val Ile Gly Ala Ile Val Val Gly LeuCys Leu Met Gly Met Gly 385 390 395 400 Val Tyr Lys Ile Arg Leu Arg CysGln Ser Ser Gly Tyr Gln Arg Ile 405 410 415 <210> SEQ ID NO 2 <211>LENGTH: 1248 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<223> OTHER INFORMATION: human normal lung cDNA library <400> SEQUENCE:2 atgccccggc agctcagcgc ggcggccgcg ctcttcgcgt ccctggccgt aattttgcac 60gatggcagtc aaatgagagc aaaagcattt ccagaaacca gagattattc tcaacctact 120gcagcagcaa cagtacagga cataaaaaaa cctgtccagc aaccagctaa gcaagcacct 180caccaaactt tagcagcaag attcatggat ggtcatatca cctttcaaac agcggccaca 240gtaaaaattc caacaactac cccagcaact acaaaaaaca ctgcaaccac cagcccaatt 300acctacaccc tggtcacaac ccaggccaca cccaacaact cacacacagc tcctccagtt 360actgaagtta cagtcggccc tagcttagcc ccttattcac tgccacccac catcacccca 420ccagctcata cagctggaac cagttcatca accgtcagcc acacaactgg gaacaccact 480caacccagta accagaccac ccttccagca actttatcga tagcactgca caaaagcaca 540accggtcaga agcctgatca acccacccat gccccaggaa caacggcagc tgcccacaat 600accacccgca cagctgcacc tgcctccacg gttcctgggc ccacccttgc acctcagcca 660tcgtcagtca agactggaat ttatcaggtt ctaaacggaa gcagactctg tataaaagca 720gagatgggga tacagctgat tgttcaagac aaggagtcgg ttttttcacc tcggagatac 780ttcaacatcg accccaacgc aacgcaagcc tctgggaact gtggcacccg aaaatccaac 840cttctgttga attttcaggg cggatttgtg aatctcacat ttaccaagga tgaagaatca 900tattatatca gtgaagtggg agcctatttg accgtctcag atccagagac agtttaccaa 960ggaatcaaac atgcggtggt gatgttccag acagcagtcg ggcattcctt caagtgcgtg 1020agtgaacaga gcctccagtt gtcagcccac ctgcaggtga aaacaaccga tgtccaactt 1080caagcctttg attttgaaga tgaccacttt ggaaatgtgg atgagtgctc gtctgactac 1140acaattgtgc ttcctgtgat tggggccatc gtggttggtc tctgccttat gggtatgggt 1200gtctataaaa tccgcctaag gtgtcaatca tctggatacc agagaatc 1248 <210> SEQ IDNO 3 <211> LENGTH: 3198 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: human normal lung cDNA library<221> NAME/KEY: CDS <222> LOCATION: (64)..(1311) <400> SEQUENCE: 3ggcaccgatt cggggcctgc ccggacttcg ccgcacgctg cagaacctcg cccagcgccc 60 accatg ccc cgg cag ctc agc gcg gcg gcc gcg ctc ttc gcg tcc ctg 108 Met ProArg Gln Leu Ser Ala Ala Ala Ala Leu Phe Ala Ser Leu 1 5 10 15 gcc gtaatt ttg cac gat ggc agt caa atg aga gca aaa gca ttt cca 156 Ala Val IleLeu His Asp Gly Ser Gln Met Arg Ala Lys Ala Phe Pro 20 25 30 gaa acc agagat tat tct caa cct act gca gca gca aca gta cag gac 204 Glu Thr Arg AspTyr Ser Gln Pro Thr Ala Ala Ala Thr Val Gln Asp 35 40 45 ata aaa aaa cctgtc cag caa cca gct aag caa gca cct cac caa act 252 Ile Lys Lys Pro ValGln Gln Pro Ala Lys Gln Ala Pro His Gln Thr 50 55 60 tta gca gca aga ttcatg gat ggt cat atc acc ttt caa aca gcg gcc 300 Leu Ala Ala Arg Phe MetAsp Gly His Ile Thr Phe Gln Thr Ala Ala 65 70 75 aca gta aaa att cca acaact acc cca gca act aca aaa aac act gca 348 Thr Val Lys Ile Pro Thr ThrThr Pro Ala Thr Thr Lys Asn Thr Ala 80 85 90 95 acc acc agc cca att acctac acc ctg gtc aca acc cag gcc aca ccc 396 Thr Thr Ser Pro Ile Thr TyrThr Leu Val Thr Thr Gln Ala Thr Pro 100 105 110 aac aac tca cac aca gctcct cca gtt act gaa gtt aca gtc ggc cct 444 Asn Asn Ser His Thr Ala ProPro Val Thr Glu Val Thr Val Gly Pro 115 120 125 agc tta gcc cct tat tcactg cca ccc acc atc acc cca cca gct cat 492 Ser Leu Ala Pro Tyr Ser LeuPro Pro Thr Ile Thr Pro Pro Ala His 130 135 140 aca gct gga acc agt tcatca acc gtc agc cac aca act ggg aac acc 540 Thr Ala Gly Thr Ser Ser SerThr Val Ser His Thr Thr Gly Asn Thr 145 150 155 act caa ccc agt aac cagacc acc ctt cca gca act tta tcg ata gca 588 Thr Gln Pro Ser Asn Gln ThrThr Leu Pro Ala Thr Leu Ser Ile Ala 160 165 170 175 ctg cac aaa agc acaacc ggt cag aag cct gat caa ccc acc cat gcc 636 Leu His Lys Ser Thr ThrGly Gln Lys Pro Asp Gln Pro Thr His Ala 180 185 190 cca gga aca acg gcagct gcc cac aat acc acc cgc aca gct gca cct 684 Pro Gly Thr Thr Ala AlaAla His Asn Thr Thr Arg Thr Ala Ala Pro 195 200 205 gcc tcc acg gtt cctggg ccc acc ctt gca cct cag cca tcg tca gtc 732 Ala Ser Thr Val Pro GlyPro Thr Leu Ala Pro Gln Pro Ser Ser Val 210 215 220 aag act gga att tatcag gtt cta aac gga agc aga ctc tgt ata aaa 780 Lys Thr Gly Ile Tyr GlnVal Leu Asn Gly Ser Arg Leu Cys Ile Lys 225 230 235 gca gag atg ggg atacag ctg att gtt caa gac aag gag tcg gtt ttt 828 Ala Glu Met Gly Ile GlnLeu Ile Val Gln Asp Lys Glu Ser Val Phe 240 245 250 255 tca cct cgg agatac ttc aac atc gac ccc aac gca acg caa gcc tct 876 Ser Pro Arg Arg TyrPhe Asn Ile Asp Pro Asn Ala Thr Gln Ala Ser 260 265 270 ggg aac tgt ggcacc cga aaa tcc aac ctt ctg ttg aat ttt cag ggc 924 Gly Asn Cys Gly ThrArg Lys Ser Asn Leu Leu Leu Asn Phe Gln Gly 275 280 285 gga ttt gtg aatctc aca ttt acc aag gat gaa gaa tca tat tat atc 972 Gly Phe Val Asn LeuThr Phe Thr Lys Asp Glu Glu Ser Tyr Tyr Ile 290 295 300 agt gaa gtg ggagcc tat ttg acc gtc tca gat cca gag aca gtt tac 1020 Ser Glu Val Gly AlaTyr Leu Thr Val Ser Asp Pro Glu Thr Val Tyr 305 310 315 caa gga atc aaacat gcg gtg gtg atg ttc cag aca gca gtc ggg cat 1068 Gln Gly Ile Lys HisAla Val Val Met Phe Gln Thr Ala Val Gly His 320 325 330 335 tcc ttc aagtgc gtg agt gaa cag agc ctc cag ttg tca gcc cac ctg 1116 Ser Phe Lys CysVal Ser Glu Gln Ser Leu Gln Leu Ser Ala His Leu 340 345 350 cag gtg aaaaca acc gat gtc caa ctt caa gcc ttt gat ttt gaa gat 1164 Gln Val Lys ThrThr Asp Val Gln Leu Gln Ala Phe Asp Phe Glu Asp 355 360 365 gac cac tttgga aat gtg gat gag tgc tcg tct gac tac aca att gtg 1212 Asp His Phe GlyAsn Val Asp Glu Cys Ser Ser Asp Tyr Thr Ile Val 370 375 380 ctt cct gtgatt ggg gcc atc gtg gtt ggt ctc tgc ctt atg ggt atg 1260 Leu Pro Val IleGly Ala Ile Val Val Gly Leu Cys Leu Met Gly Met 385 390 395 ggt gtc tataaa atc cgc cta agg tgt caa tca tct gga tac cag aga 1308 Gly Val Tyr LysIle Arg Leu Arg Cys Gln Ser Ser Gly Tyr Gln Arg 400 405 410 415 atctaattgttgc ccggggggaa tgaaaataat ggaatttaga gaactctttc 1361 Ileatcccttcca ggatggatgt tgggaaattc cctcagagtg tgggtccttc aaacaatgta 1421aaccaccatc ttctattcaa atgaagtgag tcatgtgtga tttaagttca ggcagcacat 1481caatttctaa atactttttg tttattttat gaaagatata gtgagctgtt tattttctag 1541tttcctttag aatattttag ccactcaaag tcaacatttg agatatgttg aattaacata 1601atatatgtaa agtagaataa gccttcaaat tataaaccaa gggtcaattg taactaatac 1661tactgtgtgt gcattgaaga ttttatttta cccttgatct taacaaagcc tttgctttgt 1721tatcaaatgg actttcagtg cttttactat ctgtgtttta tggtttcatg taacatacat 1781attcctggtg tagcacttaa ctccttttcc actttaaatt tgtttttgtt ttttgagacg 1841gagtttcact cttgtcaccc aggctggagt acagtggcac gatctcggct tatggcaacc 1901tccgcctccc gggttcaagt gattctcctg cttcagcttc ccgagtagct gggattacag 1961gcacacacta ccacgcctgg ctaatttttg tatttttatt atagacgggt ttcaccatgt 2021tggccagact ggtcttgaac tcttgacctc aggtgatcca cccacctcag cctcccaaag 2081tgctgggatt acaggcatga gccattgcgc ccggccttaa atgttttttt taatcatcaa 2141aaagaacaac atatctcagg ttgtctaagt gtttttatgt aaaaccaaca aaaagaacaa 2201atcagcttat attttttatc ttgatgactc ctgctccaga atgctagact aagaattagg 2261tggctacaga tggtagaact aaacaataag caagagacaa taataatggc ccttaattat 2321taacaaagtg ccagagtcta ggctaagcac tttatctata tctcatttca ttctcacaac 2381ttataagtga atgagtaaac tgagacttaa gggaactgaa tcacttaaat gtcacctggc 2441taactgatgg cagagccaga gcttgaattc atgttggtct gacatcaagg tctttggtct 2501tctccctaca ccaagttacc tacaagaaca atgacaccac actctgcctg aaggctcaca 2561cctcatacca gcatacgctc accttacagg gaaatgggtt tatccaggat catgagacat 2621tagggtagat gaaaggagag ctttgcagat aacaaaatag cctatcctta ataaatcctc 2681cactctctgg aaggagactg aggggctttg taaaacatta gtcagttgct catttttatg 2741ggattgctta gctgggctgt aaagatgaag gcatcaaata aactcaaagt atttttaaat 2801ttttttgata atagagaaac ttcgctaacc aactgttctt tcttgagtgt atagccccat 2861cttgtggtaa cttgctgctt ctgcacttca tatccatatt tcctattgtt cactttattc 2921tgtagagcag cctgccaaga attttatttc tgctgttttt tttgctgcta aagaaaggaa 2981ctaagtcagg atgttaacag aaaagtccac ataaccctag aattcttagt caaggaataa 3041ttcaagtcag cctagagacc atgttgactt tcctcatgtg tttccttatg actcagtaag 3101ttggcaaggt cctgacttta gtcttaataa aacattgaat tgtagtaaag gtttttgcaa 3161taaaaactta ctttggaaaa aaaaaaaaaa aaaaaaa 3198 <210> SEQ ID NO 4 <211>LENGTH: 416 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHERINFORMATION: human normal lung cDNA library <400> SEQUENCE: 4 Met ProArg Gln Leu Ser Ala Ala Ala Ala Leu Phe Ala Ser Leu Ala 1 5 10 15 ValIle Leu His Asp Gly Ser Gln Met Arg Ala Lys Ala Phe Pro Glu 20 25 30 ThrArg Asp Tyr Ser Gln Pro Thr Ala Ala Ala Thr Val Gln Asp Ile 35 40 45 LysLys Pro Val Gln Gln Pro Ala Lys Gln Ala Pro His Gln Thr Leu 50 55 60 AlaAla Arg Phe Met Asp Gly His Ile Thr Phe Gln Thr Ala Ala Thr 65 70 75 80Val Lys Ile Pro Thr Thr Thr Pro Ala Thr Thr Lys Asn Thr Ala Thr 85 90 95Thr Ser Pro Ile Thr Tyr Thr Leu Val Thr Thr Gln Ala Thr Pro Asn 100 105110 Asn Ser His Thr Ala Pro Pro Val Thr Glu Val Thr Val Gly Pro Ser 115120 125 Leu Ala Pro Tyr Ser Leu Pro Pro Thr Ile Thr Pro Pro Ala His Thr130 135 140 Ala Gly Thr Ser Ser Ser Thr Val Ser His Thr Thr Gly Asn ThrThr 145 150 155 160 Gln Pro Ser Asn Gln Thr Thr Leu Pro Ala Thr Leu SerIle Ala Leu 165 170 175 His Lys Ser Thr Thr Gly Gln Lys Pro Asp Gln ProThr His Ala Pro 180 185 190 Gly Thr Thr Ala Ala Ala His Asn Thr Thr ArgThr Ala Ala Pro Ala 195 200 205 Ser Thr Val Pro Gly Pro Thr Leu Ala ProGln Pro Ser Ser Val Lys 210 215 220 Thr Gly Ile Tyr Gln Val Leu Asn GlySer Arg Leu Cys Ile Lys Ala 225 230 235 240 Glu Met Gly Ile Gln Leu IleVal Gln Asp Lys Glu Ser Val Phe Ser 245 250 255 Pro Arg Arg Tyr Phe AsnIle Asp Pro Asn Ala Thr Gln Ala Ser Gly 260 265 270 Asn Cys Gly Thr ArgLys Ser Asn Leu Leu Leu Asn Phe Gln Gly Gly 275 280 285 Phe Val Asn LeuThr Phe Thr Lys Asp Glu Glu Ser Tyr Tyr Ile Ser 290 295 300 Glu Val GlyAla Tyr Leu Thr Val Ser Asp Pro Glu Thr Val Tyr Gln 305 310 315 320 GlyIle Lys His Ala Val Val Met Phe Gln Thr Ala Val Gly His Ser 325 330 335Phe Lys Cys Val Ser Glu Gln Ser Leu Gln Leu Ser Ala His Leu Gln 340 345350 Val Lys Thr Thr Asp Val Gln Leu Gln Ala Phe Asp Phe Glu Asp Asp 355360 365 His Phe Gly Asn Val Asp Glu Cys Ser Ser Asp Tyr Thr Ile Val Leu370 375 380 Pro Val Ile Gly Ala Ile Val Val Gly Leu Cys Leu Met Gly MetGly 385 390 395 400 Val Tyr Lys Ile Arg Leu Arg Cys Gln Ser Ser Gly TyrGln Arg Ile 405 410 415 <210> SEQ ID NO 5 <211> LENGTH: 280 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:human embryonic brain cDNA library <400> SEQUENCE: 5 Met Leu Gly Gln GlnGln Gln Gln Leu Tyr Ser Ser Ala Ala Leu Leu 1 5 10 15 Thr Gly Glu ArgSer Arg Leu Leu Thr Cys Tyr Val Gln Asp Tyr Leu 20 25 30 Glu Cys Val GluSer Leu Pro His Asp Met Gln Arg Asn Val Ser Val 35 40 45 Leu Arg Glu LeuAsp Asn Lys Tyr Gln Glu Thr Leu Lys Glu Ile Asp 50 55 60 Asp Val Tyr GluLys Tyr Lys Lys Glu Asp Asp Leu Asn Gln Lys Lys 65 70 75 80 Arg Leu GlnGln Leu Leu Gln Arg Ala Leu Ile Asn Ser Gln Glu Leu 85 90 95 Gly Asp GluLys Ile Gln Ile Val Thr Gln Met Leu Glu Leu Val Glu 100 105 110 Asn ArgAla Arg Gln Met Glu Leu His Ser Gln Cys Phe Gln Asp Pro 115 120 125 AlaGlu Ser Glu Arg Ala Ser Asp Lys Ala Lys Met Asp Ser Ser Gln 130 135 140Pro Glu Arg Ser Ser Arg Arg Pro Arg Arg Gln Arg Thr Ser Glu Ser 145 150155 160 Arg Asp Leu Cys His Met Ala Asn Gly Ile Glu Asp Cys Asp Asp Gln165 170 175 Pro Pro Lys Glu Lys Lys Ser Lys Ser Ala Lys Lys Lys Lys ArgSer 180 185 190 Lys Ala Lys Gln Glu Arg Glu Ala Ser Pro Val Glu Phe AlaIle Asp 195 200 205 Pro Asn Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val SerTyr Gly Glu 210 215 220 Met Ile Gly Cys Asp Asn Glu Gln Cys Pro Ile GluTrp Phe His Phe 225 230 235 240 Ser Cys Val Ser Leu Thr Tyr Lys Pro LysGly Lys Trp Tyr Cys Pro 245 250 255 Lys Cys Arg Gly Asp Asn Glu Lys ThrMet Asp Lys Ser Thr Glu Lys 260 265 270 Thr Lys Lys Asp Arg Arg Ser Arg275 280 <210> SEQ ID NO 6 <211> LENGTH: 840 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: humanembryonic brain cDNA library <400> SEQUENCE: 6 atgttagggc agcagcagcagcaactgtac tcgtcggccg cgctcctgac cggggagcgg 60 agccggctgc tcacctgctacgtgcaggac taccttgagt gcgtggagtc gctgccccac 120 gacatgcaga ggaacgtgtctgtgctgcga gagctggaca acaaatatca agaaacgtta 180 aaggaaattg atgatgtctacgaaaaatat aagaaagaag atgatttaaa ccagaagaaa 240 cgtctacagc agcttctccagagagcacta attaatagtc aagaattggg agatgaaaaa 300 atacagattg ttacacaaatgctcgaattg gtggaaaatc gggcaagaca aatggagtta 360 cactcacagt gtttccaagatcctgctgaa agtgaacgag cctcagataa agcaaagatg 420 gattccagcc aaccagaaagatcttcaaga agaccccgca ggcagcggac cagtgaaagc 480 cgtgatttat gtcacatggcaaatgggatt gaagactgtg atgatcagcc acctaaagaa 540 aagaaatcca agtcagcaaagaaaaagaaa cgctccaagg ccaagcagga aagggaagct 600 tcacctgttg agtttgcaatagatcctaat gaacctacat actgcttatg caaccaagtg 660 tcttatgggg agatgataggatgtgacaat gaacagtgtc caattgaatg gtttcacttt 720 tcatgtgttt cacttacctataaaccaaag gggaaatggt attgcccaaa gtgcagggga 780 gataatgaga aaacaatggacaaaagtact gaaaagacaa aaaaggatag aagatcgagg 840 <210> SEQ ID NO 7 <211>LENGTH: 1078 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<223> OTHER INFORMATION: human embryonic brain cDNA library <221>NAME/KEY: CDS <222> LOCATION: (92)..(931) <400> SEQUENCE: 7 tccaagctgagctgagggcc cgcggcggcc gcggccggtg catgtgcggc tgctggatgc 60 ggaggcggcggcgacggcgc ggatcggcag g atg tta ggg cag cag cag cag 112 Met Leu Gly GlnGln Gln Gln 1 5 caa ctg tac tcg tcg gcc gcg ctc ctg acc ggg gag cgg agccgg ctg 160 Gln Leu Tyr Ser Ser Ala Ala Leu Leu Thr Gly Glu Arg Ser ArgLeu 10 15 20 ctc acc tgc tac gtg cag gac tac ctt gag tgc gtg gag tcg ctgccc 208 Leu Thr Cys Tyr Val Gln Asp Tyr Leu Glu Cys Val Glu Ser Leu Pro25 30 35 cac gac atg cag agg aac gtg tct gtg ctg cga gag ctg gac aac aaa256 His Asp Met Gln Arg Asn Val Ser Val Leu Arg Glu Leu Asp Asn Lys 4045 50 55 tat caa gaa acg tta aag gaa att gat gat gtc tac gaa aaa tat aag304 Tyr Gln Glu Thr Leu Lys Glu Ile Asp Asp Val Tyr Glu Lys Tyr Lys 6065 70 aaa gaa gat gat tta aac cag aag aaa cgt cta cag cag ctt ctc cag352 Lys Glu Asp Asp Leu Asn Gln Lys Lys Arg Leu Gln Gln Leu Leu Gln 7580 85 aga gca cta att aat agt caa gaa ttg gga gat gaa aaa ata cag att400 Arg Ala Leu Ile Asn Ser Gln Glu Leu Gly Asp Glu Lys Ile Gln Ile 9095 100 gtt aca caa atg ctc gaa ttg gtg gaa aat cgg gca aga caa atg gag448 Val Thr Gln Met Leu Glu Leu Val Glu Asn Arg Ala Arg Gln Met Glu 105110 115 tta cac tca cag tgt ttc caa gat cct gct gaa agt gaa cga gcc tca496 Leu His Ser Gln Cys Phe Gln Asp Pro Ala Glu Ser Glu Arg Ala Ser 120125 130 135 gat aaa gca aag atg gat tcc agc caa cca gaa aga tct tca agaaga 544 Asp Lys Ala Lys Met Asp Ser Ser Gln Pro Glu Arg Ser Ser Arg Arg140 145 150 ccc cgc agg cag cgg acc agt gaa agc cgt gat tta tgt cac atggca 592 Pro Arg Arg Gln Arg Thr Ser Glu Ser Arg Asp Leu Cys His Met Ala155 160 165 aat ggg att gaa gac tgt gat gat cag cca cct aaa gaa aag aaatcc 640 Asn Gly Ile Glu Asp Cys Asp Asp Gln Pro Pro Lys Glu Lys Lys Ser170 175 180 aag tca gca aag aaa aag aaa cgc tcc aag gcc aag cag gaa agggaa 688 Lys Ser Ala Lys Lys Lys Lys Arg Ser Lys Ala Lys Gln Glu Arg Glu185 190 195 gct tca cct gtt gag ttt gca ata gat cct aat gaa cct aca tactgc 736 Ala Ser Pro Val Glu Phe Ala Ile Asp Pro Asn Glu Pro Thr Tyr Cys200 205 210 215 tta tgc aac caa gtg tct tat ggg gag atg ata gga tgt gacaat gaa 784 Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile Gly Cys Asp AsnGlu 220 225 230 cag tgt cca att gaa tgg ttt cac ttt tca tgt gtt tca cttacc tat 832 Gln Cys Pro Ile Glu Trp Phe His Phe Ser Cys Val Ser Leu ThrTyr 235 240 245 aaa cca aag ggg aaa tgg tat tgc cca aag tgc agg gga gataat gag 880 Lys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cys Arg Gly Asp AsnGlu 250 255 260 aaa aca atg gac aaa agt act gaa aag aca aaa aag gat agaaga tcg 928 Lys Thr Met Asp Lys Ser Thr Glu Lys Thr Lys Lys Asp Arg ArgSer 265 270 275 agg tagtaaaggc catccacatt ttaaagggtt atttgactattatataatcc 981 Arg 280 gtttgctttc agaaaatgtt ttagggtaaa tgcataagactatgcaataa ttattaatca 1041 ttagtattaa tggtgtatta aaagttgttg tactttg 1078<210> SEQ ID NO 8 <211> LENGTH: 280 <212> TYPE: PRT <213> ORGANISM: Homosapiens <223> OTHER INFORMATION: human embryonic brain cDNA library<400> SEQUENCE: 8 Met Leu Gly Gln Gln Gln Gln Gln Leu Tyr Ser Ser AlaAla Leu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu Thr Cys Tyr ValGln Asp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His Asp Met Gln ArgAsn Val Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr Gln Glu Thr LeuLys Glu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys Glu Asp Asp LeuAsn Gln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln Arg Ala Leu IleAsn Ser Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile Val Thr Gln MetLeu Glu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met Glu Leu His SerGln Cys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg Ala Ser Asp LysAla Lys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser Ser Arg Arg ProArg Arg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp Leu Cys His MetAla Asn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro Pro Lys Glu LysLys Ser Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 Lys Ala Lys GlnGlu Arg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205 Pro Asn GluPro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215 220 Met IleGly Cys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225 230 235 240Ser Cys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr Cys Pro 245 250255 Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser Thr Glu Lys 260265 270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 <210> SEQ ID NO 9 <211>LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:PCR primer sequence for TSC403 <400> SEQUENCE: 9 gatctgacac 10 <210> SEQID NO 10 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: PCR primer P1 <400> SEQUENCE: 10 gatcggatccaggaggatgc gggtccgg 28 <210> SEQ ID NO 11 <211> LENGTH: 30 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: PCR primer P2 <400>SEQUENCE: 11 gatcctcgag ttactgtggt ggctgctgct 30

1. A gene having a nucleotide sequence coding for the amino acidsequence shown in SEQ ID NO:1.
 2. A gene which is a polynucleotideselected from among the following (a), (b) and (c): (a) a polynucleotidecontaining the nucleotide sequence shown in SEQ ID NO:2 or acomplementary sequence thereto, (b) a polynucleotide that hybridizes tosaid polynucleotide (a) under stringent conditions, and (c) apolynucleotide having at least 95% homology to a polynucleotide codingfor a polypeptide containing the amino acid sequence shown in SEQ IDNO:1.
 3. The gene defined in claim 1 or 2 which is a human gene.
 4. Thegene defined in claim 2 which has the nucleotide sequence shown in SEQID NO:3.
 5. An oligonucleotide having a sequence consisting of at least15 consecutive nucleotides in the nucleotide sequence shown in SEQ IDNO:2.
 6. A probe comprising a sequence consisting of at least 15consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO:2.7. A primer comprising a sequence consisting of at least 15 consecutivenucleotides in the nucleotide sequence shown in SEQ ID NO:2.
 8. A methodfor diagnosis of cancer which comprises detecting expression of the genedefined in claim 1 using a probe having a sequence consisting of atleast 15 consecutive nucleotides in the nucleotide sequence shown in SEQID NO:2 or a primer having a sequence consisting of at least 15consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO:2.9. The method of diagnosis defined in claim 8 wherein the cancer is amember selected from among cancer of the mammary gland, cancer of theuterine tube, cancer of the esophagus, cancer of the colon, cancer ofthe rectum, cancer of the thyroid gland, cancer of the parotid gland,cancer of the ureter, cancer of the ovary and cancer of the pancreas.10. A diagnostic kit for cancer which comprises a probe having asequence consisting of at least 15 consecutive nucleotides in thenucleotide sequence shown in SEQ ID NO:2 or a primer having a sequenceconsisting of at least 15 consecutive nucleotides in the nucleotidesequence shown in SEQ ID NO:2 as an essential component.
 11. A proteinhaving an amino acid sequence shown in SEQ ID NO:1 which is encoded bythe gene defined in claim
 1. 12. An antibody having a binding affinityfor the protein defined in claim
 11. 13. A human gene comprising anucleotide sequence coding for the amino acid sequence shown in SEQ IDNO:4.
 14. A gene which is a polynucleotide selected from among thefollowing (a), (b) and (c): (a) a polynucleotide containing a nucleotidesequence shown in SEQ ID NO:5 or a complementary sequence thereto, (b) apolynucleotide that hybridizes to said polynucleotide (a) understringent conditions, and (c) a polynucleotide having at least 95%homology to a polynucleotide coding for a polypeptide containing theamino acid sequence shown in SEQ ID NO:4.
 15. The gene defined in claim14 which is a human gene having the nucleotide sequence shown in SEQ IDNO:6.
 16. An oligonucleotide having a sequence consisting of at least 15consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO:5.17. A probe having a sequence consisting of at least 15 consecutivenucleotides in the nucleotide sequence shown in SEQ ID NO:5.
 18. Aprimer having a sequence consisting of at least 15 consecutivenucleotides in the nucleotide sequence shown in SEQ ID NO:5.
 19. Amethod for diagnosis of cancer which comprises detecting expression ofthe gene defined in claim 13 using a probe having a sequence consistingof at least 15 consecutive nucleotides in the nucleotide sequence shownin SEQ ID NO:5 or a primer having a sequence consisting of at least 15consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO:5.20. The method of diagnosis defined in claim 19 wherein the cancer is amember selected from among colorectal cancer, cancer of the esophagus,cancer of the uterine tube and cancer of the stomach.
 21. A diagnostickit for cancer which comprises a probe having a sequence consisting ofat least 15 consecutive nucleotides in the nucleotide sequence shown inSEQ ID NO:5 or a primer having a sequence consisting of at least 15consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO:2as an essential component.
 22. A protein having an amino acid sequenceof SEQ ID NO:4 which is encoded by the gene defined in claim
 13. 23. Anantibody having a binding affinity for the protein defined in claim 22.